Subcutaneous administration of ADAMTS13

ABSTRACT

This invention relates to methods of subcutaneous administration of ADAMTS13 formulations to a treat a disease or condition associated with ADAMTS13 and VWF dysfunction. Furthermore, evidence of the unexpectedly high bioavailability of ADAMTS13 formulations administered subcutaneously is provided herein.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 61/794,659 filed Mar. 15, 2013, the disclosure of which ishereby incorporated herein by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin typeI motifs) proteins are a family of metalloproteinases containing numberof conserved domains, including a zinc-dependant catalytic domain, acystein-rich domain, a disintegrin-like domain, and at least one, and inmost cases multiple, thrombospondin type I repeats (for review, seeNicholson et al., BMC Evol Biol. 2005 Feb. 4; 5(1):11). These proteins,which are evolutionarily related to the ADAM and MMP families ofmetalloproteinases (Jones G C, Curr Pharm Biotechnol. 2006 February;7(1):25-31), are secreted enzymes that have been linked to a number ofdiseases and conditions including thrombotic thrombocytopenic purpura(TTP) (Moake J L, Semin Hematol. 2004 January; 41(1):4-14), connectivetissue disorders, cancers, inflammation (Nicholson et al.), and severeplasmodium falciparum malaria (Larkin et al., PLoS Pathog. 2009 March;5(3):e1000349). Because of these associations, the ADAMTS enzymes havebeen recognized as potential therapeutic targets for a number ofpathologies (Jones G C, Curr Pharm Biotechnol. 2006 February;7(1):25-31).

One ADAMTS family member, ADAMTS13, cleaves von Willebrand factor (vWF)between residues Tyr 1605 and Met 1606. Loss of ADAMTS13 activity hasbeen linked to a number of conditions, such as TTP (Moake J L, SeminHematol. 2004 January; 41(1):4-14), acute and chronic inflammation(Chauhan et al., J Exp Med. 2008 Sep. 1; 205(9):2065-74), and mostrecently, severe plasmodium falciparum malaria (Larkin et al., PLoSPathog. 2009 March; 5(3):e1000349).

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized bythrombotic microangiopathy, thrombocytopenia and microvascularthrombosis that can cause various degrees of tissue ischemia andinfarction. Clinically, TTP patients are diagnosed by symptoms such asthrombocytopenia, schistocytes (fragments of erythrocytes) and elevatedlevels of lactate dehydrogenase (Moake J L. Thromboticmicroangiopathies. N Engl J. Med. 2002; 347:589-600; Moake J L. vonWillebrand factor, ADAMTS-13, and thrombotic thrombocytopenic purpura.Semin Hematol. 2004; 41:4-14; Sadler J E, Moake J L, Miyata T, George JN. Recent advances in thrombotic thrombocytopenic purpura. Hematology(Am Soc Hematol Educ Program). 2004: 407-423; Sadler J E. New conceptsin von Willebrand disease. Annu Rev Med. 2005; 56:173-191).

There are two major types of TTP: acquired (noninherited/idopathic) andfamilial (inherited) (Tsai H M, Lian E C. Antibodies to von Willebrandfactor-cleaving protease in acute thrombotic thrombocytopenic purpura. NEngl J. Med. 1998; 339:1585-1594; Furlan M, Lammle B. Deficiency of vonWillebrand factor-cleaving protease in familial and acquired thromboticthrombocytopenic purpura. Baillieres Clin Haematol. 1998; 11:509-514).Genetic mutations in the ADAMTS13 gene cause the familial form of TTPwhereas people with acquired TTP do not have the mutations. Rather,acquired TTP is characterized by the production of specific antibodies.

In 1982, Moake et al. found unusually large von Willebrand factor(UL-vWF) multimers in the plasma of the patients with chronic relapsingTTP (Moake J L, Rudy C K, Troll J H, Weinstein M J, Colannino N M,Azocar J, Seder R H, Hong S L, Deykin D. Unusually large plasma factorVIII: von Willebrand factor multimers in chronic relapsing thromboticthrombocytopenic purpura. N Engl J. Med. 1982; 307:1432-1435). The linkbetween UL-vWF and TTP gained support with independent findings byFurlan et al. and Tsai and Lian that most patients suffering from TTPare deficient in a plasma metalloprotease, now known to be ADAMTS13,that cleaves vWF (Furlan M, Robles R, Solenthaler M, Wassmer M, SandozP, Laemmle B. Deficient activity of von Willebrand factor-cleavingprotease in chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1997; 89:3097-3103; Tsai H M, Sussman, I I, Ginsburg D, LankhofH, Sixma J J, Nagel R L. Proteolytic cleavage of recombinant type 2A vonWillebrand factor mutants R834W and R834Q: inhibition by doxycycline andby monoclonal antibody VP-1. Blood. 1997; 89:1954-1962; Tsai H M, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acutethrombotic thrombocytopenic purpura. N Engl J. Med. 1998;339:1585-1594).

The ADAMTS13 protease is a 190 kDa glycosylated protein producedpredominantly by the liver (Levy G G, Nichols W C, Lian E C, Foroud T,McClintick J N, McGee B M, Yang A Y, Siemieniak D R, Stark K R, GruppoR, Sarode R, Shurin S B, Chandrasekaran V, Stabler S P, Sabio H,Bouhassira E E, Upshaw J D, Jr., Ginsburg D, Tsai H M. Mutations in amember of the ADAMTS gene family cause thrombotic thrombocytopenicpurpura. Nature. 2001; 413:488-494; Fujikawa K, Suzuki H, McMullen B,Chung D. Purification of human von Willebrand factor-cleaving proteaseand its identification as a new member of the metalloproteinase family.Blood. 2001; 98:1662-1666; Zheng X, Chung D, Takayama T K, Majerus E M,Sadler J E, Fujikawa K. Structure of von Willebrand factor-cleavingprotease (ADAMTS13), a metalloprotease involved in thromboticthrombocytopenic purpura. J Biol. Chem. 2001; 276:41059-41063; SoejimaK, Mimura N, Hirashima M, Maeda H, Hamamoto T, Nakagaki T, Nozaki C. Anovel human metalloprotease synthesized in the liver and secreted intothe blood: possibly, the von Willebrand factor-cleaving protease; JBiochem (Tokyo). 2001; 130:475-480; Gerritsen H E, Robles R, Lammle B,Furlan M. Partial amino acid sequence of purified von Willebrandfactor-cleaving protease. Blood. 2001; 98:1654-1661).

Mutations in the ADAMTS13 gene have been shown to cause TTP (Levy G G,Nichols W C, Lian E C, Foroud T, McClintick J N, McGee B M, Yang A Y,Siemieniak D R, Stark K R, Gruppo R, Sarode R, Shurin S B,Chandrasekaran V, Stabler S P, Sabio H, Bouhassira E E, Upshaw J D, Jr.,Ginsburg D, Tsai H M. Mutations in a member of the ADAMTS gene familycause thrombotic thrombocytopenic purpura. Nature. 2001; 413:488-494).Idiopathic TTP, often caused by autoantibodies inhibiting ADAMTS-13activity, is a more common disorder that occurs in adults and olderchildren and can recur at regular intervals in 11-36% of patients (TsaiH M, Lian E C. Antibodies to von Willebrand factor-cleaving protease inacute thrombotic thrombocytopenic purpura. N Engl J. Med. 1998;339:1585-1594; Furlan M, Lammle B. Deficiency of von Willebrandfactor-cleaving protease in familial and acquired thromboticthrombocytopenic purpura. Baillieres Clin Haematol. 1998; 11:509-514).

Non neutralizing autoantibodies could also inhibit ADAMTS activity byinducing clearance from circulation (Scheiflinger F, Knobl P, TrattnerB, Plaimauer B, Mohr G, Dockal M, Dorner F, Rieger M. NormeutralizingIgM and IgG antibodies to von Willebrand factor-cleaving protease(ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura.Blood. 2003; 102:3241-3243). Plasma ADAMTS13 activity in healthy adultsranges from 50% to 178% (Moake J L. Thrombotic thrombocytopenic purpuraand the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002;126:1430-1433). In most patients with familial or acquired TTP, plasmaADAMTS13 activity is absent or less than 5% of the normal. Withouttreatment the mortality rate exceeds 90%, but plasma therapy has reducedmortality to about 20% (Moake J L. Thrombotic thrombocytopenic purpuraand the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002;126:1430-1433).

vWF synthesized in megakaryocytes and endothelial cells is stored inplatelet—granules and Weibel-Palade bodies, respectively, as ultra largevWF (UL-vWF) (Moake J L, Rudy C K, Troll J H, Weinstein M J, Colannino NM, Azocar J, Seder R H, Hong S L, Deykin D. Unusually large plasmafactor VIII: von Willebrand factor multimers in chronic relapsingthrombotic thrombocytopenic purpura. N Engl J. Med. 1982; 307:1432-1435;Wagner D D, Olmsted J B, Marder V J. Immunolocalization of vonWillebrand protein in Weibel-Palade bodies of human endothelial cells.J. Cell Biol. 1982; 95:355-360; Wagner D D, Bonfanti R. von Willebrandfactor and the endothelium. Mayo Clin Proc. 1991; 66:621-627; Sporn L A,Marder V J, Wagner D D. von Willebrand factor released fromWeibel-Palade bodies binds more avidly to extracellular matrix than thatsecreted constitutively. Blood. 1987; 69:1531-1534; Tsai H M, Nagel R L,Hatcher V B, Sussman, I I. Endothelial cell-derived high molecularweight von Willebrand factor is converted into the plasma multimerpattern by granulocyte proteases. Biochem Biophys Res Commun. 1989;158:980-985; Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Multimericcomposition of endothelial cell-derived von Willebrand factor. Blood.1989; 73:2074-2076). Once secreted from endothelial cells, these UL-vWFmultimers are cleaved by ADAMTS13 in circulation into a series ofsmaller multimers at specific cleavage sites within the vWF molecule(Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Endothelialcell-derived high molecular weight von Willebrand factor is convertedinto the plasma multimer pattern by granulocyte proteases. BiochemBiophys Res Commun. 1989; 158:980-985; Dent J A, Galbusera M, Ruggeri ZM. Heterogeneity of plasma von Willebrand factor multimers resultingfrom proteolysis of the constituent subunit. J Clin Invest. 1991;88:774-782; Furlan M, Robles R, Affolter D, Meyer D, Baillod P, LammleB. Triplet structure of von Willebrand factor reflects proteolyticdegradation of high molecular weight multimers. Proc Natl Acad Sci USA.1993; 90:7503-7507).

ADAMTS13 cleaves at the Tyr842-Met843 bond in the central A2 domain ofthe mature vWF subunit and requires zinc or calcium for activity (Dent JA, Berkowitz S D, Ware J, Kasper C K, Ruggeri Z M. Identification of acleavage site directing the immunochemical detection of molecularabnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci USA.1990; 87:6306-6310). vWF exists in “ball-of-yarn” and filamentous formas seen by electron microscopy (Slayter H, Loscalzo J, Bockenstedt P,Handin R I. Native conformation of human von Willebrand protein.Analysis by electron microscopy and quasi-elastic light scattering. J.Biol. Chem. 1985; 260:8559-8563). Furthermore, atomic force microscopyconfirms that vWF exits in a globular conformation under staticconditions and an unfolded filamentous state after exposure to shearstress (Siedlecki C A, Lestini B J, Kottke-Marchant K K, Eppell S J,Wilson D L, Marchant R E. Shear-dependent changes in thethree-dimensional structure of human von Willebrand factor. Blood. 1996;88:2939-2950). This could occur also in vivo when one end of the vWFfilament is anchored to a surface.

Thrombi of TTP patients consist of little fibrin and mainly of vWF andplatelets, suggesting vWF-mediated platelet aggregation as a cause ofthrombosis (Asada Y, Sumiyoshi A, Hayashi T, Suzumiya J, Kaketani K.Immunohistochemistry of vascular lesion in thrombotic thrombocytopenicpurpura, with special reference to factor VIII related antigen. ThrombRes. 1985; 38:469-479). Patients with relapsing TTP have ultra-largemultimers in the plasma. The UL-vWF multimers accumulate over timebecause the persistence of the inhibitor (Anti-ADAMTS 13 Ab) decreasesADAMTS13 activity. The UL-vWF multimers are hyperactive and unfold as aresult of shear stress causing platelet aggregation, resulting inintravascular thrombosis (Tsai H M. Von Willebrand factor, ADAMTS13, andthrombotic thrombocytopenic purpura. J Mol. Med. 2002; 80:639-647; TsaiH M. Deficiency of ADAMTS-13 in thrombotic and thrombocytopenic purpura.J Thromb Haemost. 2003; 1:2038-2040; discussion 2040-2035).

It is believed that the presence of hyper-reactive UL-vWF multimers inthe plasma due to ADAMTS13 deficiency could be associated with anincreased risk of arterial thrombosis linked to coronary heart disease.Furthermore, ADAMTS13 has been linked to cerebral infarction, myocardialinfarction, ischemic/reperfusion injury, deep vein thrombosis, anddisseminated intravascular coagulation. Accordingly, there is a need forpharmaceutical formulations of ADAMTS13 proteins suitable for thetreatment of various diseases and conditions associated with ADAMTS13and VWF dysfunction.

However, pharmaceutical formulations comprising very large and labilemolecules such as ADAMTS13 can generally only be administeredintravenously. This is because such pharmaceutical formulations normallyexhibit a very low bioavailablity due to insufficient absorption andsevere degradation when given subcutaneously, intramuscularly, andintradermally. Accordingly, due to the low bioavailability, large andlabile proteins are normally administered intravenously to providedirect availability to the blood stream.

While ADAMTS13 can be administered intravenously to treat variousdiseases and conditions associated with ADAMTS13 and VWF dysfunction, itis inconvenient and not easy for patients to handle. Particularly,ADAMTS13 formulations are often administered regularly throughout apatient's life. For example, patients with familial (inherited) TTPbegin treatment with intravenous ADAMTS13 in their first year of life.Accordingly, it would be advantageous to subcutaneously administer apharmaceutical composition of ADAMTS13. However, low bioavailabilitiesof subcutaneously administered large and labile protein formulations hasprevented the development of such subcutaneous formulations.

Previous studies have reported that certain coagulation factors VII,VIII, and IX that are suitable for subcutaneous administration. Forexample, PCT/SE95/00348 reports a Factor VIII formulation that is highlypurified that contains additives such as hydrolyzed gelatin, hyaluronicacid, and soybean oil emulsion. The purification and additives allowedfor the Factor VIII formulation to be highly concentrated. This highlyconcentrated formulation resulted in a bioavailability of at least about15% and suitably at least about 30% after subcutaneous, intramuscular,or intradermal administration compared to the bioavailability afterintravenous administration. However, 15-30% bioavailability ofsubcutaneous administration compared to intravenous administration isstill very low and would not be effective at treating ADAMTS13disorders.

Furthermore, the prior studies do not provide a general principle forsubcutaneous administration of large and labile proteins. Rather, priorstudies present evidence of that it is subcutaneous pharmaceuticalcompositions of large and labile proteins are difficult to preparebecause the compositions lack the requisite bioavailability forsubcutaneous administration.

Described herein is a method of subcutaneously administering an ADAMTS13formulation to a treat a disease or condition associated with ADAMTS13and VWF dysfunction. Specifically, evidence of the unexpectedly highbioavailability, up to approximately 70%, of ADAMTS13 formulationsadministered subcutaneously is provided herein.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method for treating ablood clotting disorder in a mammal, the method comprisingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal in need thereof,wherein the therapeutically effective amount of ADAMTS13 comprises20-4000 activity units per kilogram.

In one embodiment of the methods provided herein, the clotting disorderis selected from the group consisting of inherited TTP, acquired TTP,cerebral infarction, myocardial infarction. ischemic/reperfusion injury,deep vein thrombosis, and sepsis-related disseminated intravascularcoagulation.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is 50-80% as compared tointravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 50% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 55% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 60% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 65% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 70% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 75% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 80% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bleeding episodeis inherited TTP.

In one embodiment of the methods provided herein, the therapeuticallyeffective amount comprises at least 20-160 activity units per kilogram.

In one embodiment of the methods provided herein, the bleeding episodeis acquired TTP.

In one embodiment of the methods provided herein, the therapeuticallyeffective amount comprises at least 40-2000 activity units per kilogram.

In one embodiment of the methods provided herein, the bleeding episodeis cerebral infarction and/or ischemia reperfusion injury.

In one embodiment of the methods provided herein, the therapeuticallyeffective amount comprises at least 40-4000 activity units per kilogram

In one embodiment of the methods provided herein, the bleeding episodeis myocardial infarction and/or ischemia reperfusion injury.

In one embodiment of the methods provided herein, the therapeuticallyeffective amount comprises at least 40-2000 activity units per kilogram.

In one embodiment of the methods provided herein, the ADAMTS13 isadministered in a single bolus injection, monthly, every two weeks,weekly, twice a week, daily, every 12 hours, every 8 hours, every sixhours, every four hours, or every two hours.

In one embodiment of the methods provided herein, the ADAMTS13 isrecombinant.

In one embodiment of the methods provided herein, the ADAMTS13 is plasmaderived.

In one embodiment of the methods provided herein, the mammal is a human.

In one embodiment of the methods provided herein, the composition is astable aqueous solution ready for administration.

In one embodiment of the methods provided herein, the composition islyophilized.

In one embodiment of the methods provided herein, the composition isreconstituted with a pharmaceutically acceptable vehicle suitable forinjection.

In one aspect, the present disclosure provides a method for treating ableeding episode in a mammal, the method comprising subcutaneouslyadministering a therapeutically effective amount of a compositioncomprising isolated ADAMTS13 to the mammal in need thereof, wherein thetherapeutically effective amount of ADAMTS13 comprises at least 120-300%of the amount of a standard intravenous dose for a specific indicationas measured in activity units per kilogram.

In one embodiment of the methods provided herein, the specificindication is inherited TTP and the standard intravenous dose is 10-80activity units per kilogram.

In one embodiment of the methods provided herein, the specificindication is acquired TTP and the standard intravenous dose is 20-1000activity units per kilogram.

In one embodiment of the methods provided herein, the specificindication is myocardial infarction and/or ischemia reperfusion injuryand the standard intravenous dose is 20-2000 activity units perkilogram.

In one embodiment of the methods provided herein, the specificindication is cerebral infarction and/or ischemia reperfusion injury andthe standard intravenous dose is 20-2000 activity units per kilogram.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is 50-80% as compared tointravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 50% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 55% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 60% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 65% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 70% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 75% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the bioavailability ofthe ADAMTS13 after subcutaneous administration is at least 80% ascompared to intravenous administration normalized for the same dose.

In one embodiment of the methods provided herein, the ADAMTS13 isadministered in a single bolus injection, monthly, every two weeks,weekly, twice a week, daily, every 12 hours, every 8 hours, every sixhours, every four hours, or every two hours.

In one embodiment of the methods provided herein, the ADAMTS13 isrecombinant.

In one embodiment of the methods provided herein, the ADAMTS13 is plasmaderived.

In one embodiment of the methods provided herein, the mammal is a human.

In one embodiment of the methods provided herein, the composition is astable aqueous solution ready for administration.

In one embodiment of the methods provided herein, the composition islyophilized.

In one embodiment of the methods provided herein, the composition isreconstituted with a pharmaceutically acceptable vehicle suitable forinjection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mean plasma concentrations of ADAMTS13 activity. MedianT_(max) after s.c. administration was 24 h for ADAMTS13 activity.

FIG. 2 shows the mean plasma concentrations of ADAMTS13 activity. MedianT_(max) after s.c. administration was 28 h for ADAMTS13 antigen

FIG. 3 shows observed (circles) and predicted concentrations (solidlines) for individual animals. Concentrations following i.v.administration were fitted using a two-compartmental model whereasconcentrations following s.c. administration were fitted using aone-compartmental model with first-order absorption and first orderelimination. Both models were modified by inclusion of an additionalcovariate to model the assumed constant endogenous ADAMTS13 activity.

FIG. 4 shows the concentrations observed adjusted for borderline valuesand corresponding predicted concentrations for individual animals.Concentrations following i.v. administration were predicted using atwo-compartmental model whereas concentrations following s.c.administration were predicted using a one-compartmental model withfirst-order absorption and first-order elimination.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

ADAMTS13 (A13) is a plasma metalloprotease which cleaves von Willebrandfactor (VWF) multimers and down regulates their activity in plateletaggregation. ADAMTS13 is associated clotting disorders such as inheritedthrombotic thrombocytopenic purpura (TTP), acquired TTP, cerebralinfarction, myocardial infarction, ischemic/reperfusion injury, deepvein thrombosis, and disseminated intravascular coagulation (DIC) suchas sepsis-related DIC.

Current treatment of these clotting disorders involves intravenousadministration of ADMTS13 formulations. Treatment is currently limitedto intravenous administration because ADAMTS13 is a large protein andlarge proteins are generally not stable in formulations with highbioavailabilities suitable for subcutaneous administration. The matureADAMTS13 has a calculated molecular mass of about 145 kDa whereaspurified plasma-derived ADAMTS13 has an apparent molecular mass of about180 kDa probably due to post-translational modifications consisting withpresent consensus sequences for 10 potential N-glycosylation sites, andseveral O-glycosylation sites and one C-mannosylation site in the TSP1repeats.

Proteins and molecules that are large and labile such as ADAMTS13, aregenerally limited to intravenous administration due to the lowbioavailability of the formulations when administered subcutaneously.For example, previous studies report that Factor VIII, a 170 to 300 kDaprotein, is typically administered intravenously because Factor VIIIformulations normally exhibit a very low bioavailability due toinsufficient absorption and severe degradation when administeredsubcutaneously, intramuscularly or intradermally. See PCT/SE95/00348.

For example, it has been reported that Factor VIII concentrate injectedintramuscularly yielded a maximum circulating level of only 1.4% of thenormal plasma level with and without sodium citrate as an additive toused to prevent degradation and increase absorption (Pool et al, NewEngland J. Medicine, vol. 275, no. 10, p. 547-548, 1966). The studiesfurther revealed that there was no significant difference in theactivity recovered in the circulation regardless of whether such citratewas added to the preparation. In a later study, a high-purity factorVIII was administered intramuscularly to haemophilic dogs and humanvolunteers (Johnson et al, Br. J. Hematology, vol. 21, p. 21-41, 1971).Although, the doses were much larger than used by Pool et al., neitherthe dogs nor the human volunteers showed a significant rise in plasmafactor VIII levels. In fact, the plasma factor VIII concentration in thehaemophilic human volunteers remained below 1% of the normal plasmalevel, i.e. the severe haemophilia A prevailed even after administrationin the absence of additives increasing the bioavailability.

There has been some success at subcutaneous delivery of small proteinssuch as Factor IX that do not degrade and aggregate like large, labileproteins such as Factor VIII and ADAMTS13. For example, subcutaneousadministration of factor IX without additives is known from Berettini etal., “Subcutaneous factor IX administration to patients with hemophiliaB,” Am. J. Hematology, 47(1):61-62, 1994. However, even Factor IX, whichis only 56 kDa, exhibited poor and very slow transport into thecirculation.

Due to the low bioavailability of proteins such as Factors VIII and IXas described above, methods of subcutaneous delivery of large and labileproteins are not generally pursued. Rather, such proteins are normallygiven intravenously so that the formulation is directly available in theblood stream. It would however be advantageous if a medicament could begiven subcutaneously because subcutaneous administration is a minimallyinvasive mode of administration. Subcutaneous administration is also themost versatile mode of administration that can be used for short termand long term therapies. Subcutaneous administration can be performed byinjection or by implantation of a sustained or timed release devicebeneath the surface of the skin. The site of the injection or device canbe rotated when multiple injections or devices are needed.

Accordingly, subcutaneous formulations much easier to handle for thepatient, especially since the formulation may have to be taken regularlyduring the whole life (e.g., starting as early as a child's first yearof life). Furthermore, the easy and speed of subcutaneous deliveryallows increased patient compliance and quicker access to medicationwhen needed. Thus, there is a benefit and need for subcutaneousformulations of ADAMTS13.

The present invention is based on the unexpected discovery of asuccessful method of subcutaneously administering liquid and lyophilizedformulations of purified ADAMTS proteins.

II. Definitions

As used herein, “ADAMTS13” or “A13” refer to a metalloprotease of theADAMTS (a disintegrin and metalloproteinase with thrombospondin type Imotifs) family that cleaves von Willebrand factor (vWF) between residuesTyr 1605 and Met 1606. In the context of the present invention, an“ADAMTS13 protein” embraces any ADAMTS13 protein, for example, ADAMTS13from a mammal such as a primate, human (NP620594), monkey, rabbit, pig,bovine (XP610784), rodent, mouse (NP001001322), rat (XP342396), hamster,gerbil, canine, feline, frog (NP001083331), chicken (XP415435), andbiologically active derivatives thereof. As used herein, “ADAMTS13proteins” refer to recombinant and plasma derived ADAMTS13 proteins.Mutant and variant ADAMTS13 proteins having activity are also embraced,as are functional fragments and fusion proteins of the ADAMTS13proteins. Furthermore, the ADAMTS13 proteins of the invention mayfurther comprise tags that facilitate purification, detection, or both.The ADAMTS13 proteins described herein may further be modified with atherapeutic moiety or a moiety suitable imaging in vitro or in vivo.

Human ADAMTS13 proteins include, without limitation, polypeptidescomprising the amino acid sequence of GenBank accession number NP 620594or a processed polypeptide thereof, for example a polypeptide in whichthe signal peptide (amino acids 1 to 29) and/or propeptide (amino acids30-74) have been removed. Many natural variants of human ADAMTS13 areknown in the art, and are embraced by the formulations of the presentinvention, some of which include mutations selected from R7W, V88M,H96D, R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H, Q448E,Q456H, P457L, P475S, C508Y, R528G, P618A, R625H, I673F, R692C, A732V,E740K, A900V, S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W,R1095W, R1123C, C1213Y, T12261, G1239V, and R1336W. Additionally,ADAMTS13 proteins include natural and recombinant proteins that havebeen mutated, for example, by one or more conservative mutations at anon-essential amino acid. Preferably, amino acids essential to theenzymatic activity of ADAMTS13 will not be mutated. These include, forexample, residues known or presumed to be essential for metal bindingsuch as residues 83, 173, 224, 228, 234, 281, and 284, and residuesfound in the active site of the enzyme, e.g., residue 225. Similarly, inthe context of the present invention, ADAMTS13 proteins includealternate isoforms, for example, isoforms lacking amino acids 275 to 305and/or 1135 to 1190 of the full-length human protein.

Likewise, ADAMTS13 proteins may be further modified, for example, bypost-translational modifications (e.g., glycosylation at one or moreamino acids selected from human residues 142, 146, 552, 579, 614, 667,707, 828, 1235, 1354, or any other natural or engineered modificationsite) or by ex vivo chemical or enzymatic modification, includingwithout limitation, glycosylation, modification by water soluble polymer(e.g., PEGylation, sialylation, HESylation, etc.), tagging, and thelike.

As used herein, “blood clotting disorder” is defined as a disorder thatincludes dysfunctional platelet recruitment as well as dysfunctionalneutrophil recruitment. Non-limiting examples of “blood clottingdisorders” include inherited thrombotic thrombocytopenic purpura (TTP),acquired TTP, cerebral infarction, myocardial infarction,ischemic/reperfusion injury, deep vein thrombosis, and disseminatedintravascular coagulation (DIC) such as sepsis-related DIC.

As used herein, “one unit of ADAMTS13 activity” is defined as the amountof activity in 1 ml of pooled normal human plasma, regardless of theassay being used. For example, one unit of ADAMTS13 FRETS-VWF73 activityis the amount of activity needed to cleave the same amount ofFRETS-VWF73 substrate (Kokame et al., Br J. Haematol. 2005 April;129(1):93-100) as is cleaved by one ml of pooled normal human plasma.Additional activity assays can also be used to determine the activity ofone unit of ADAMTS13. For example, direct ADAMTS13 activity assays canbe performed to detect the cleavage of either full-length VWF moleculesor VWF fragments using SDS agrose gel electrophoresis and indirectdetection of ADAMTS13 activity can be detected with collagen bindingassays.

As used herein, the terms “ADAMTS13” and “biologically activederivative”, respectively, also include polypeptides obtained viarecombinant DNA technology. Alternatively, ADAMTS13 can also refer tothe plasma derived ADAMTS13 purified from pooled human blood. Therecombinant ADAMTS13 (“rADAMTS13”), e.g. recombinant human ADAMTS13(“r-hu-ADAMTS13”), may be produced by any method known in the art. Onespecific example is disclosed in WO 02/42441 which is incorporatedherein by reference with respect to the method of producing recombinantADAMTS13. This may include any method known in the art for (i) theproduction of recombinant DNA by genetic engineering, e.g. via reversetranscription of RNA and/or amplification of DNA, (ii) introducingrecombinant DNA into prokaryotic or eukaryotic cells by transfection,i.e. via electroporation or microinjection, (iii) cultivating saidtransformed cells, e.g. in a continuous or batchwise manner, (iv)expressing ADAMTS13, e.g. constitutively or upon induction, and (v)isolating said ADAMTS13, e.g. from the culture medium or by harvestingthe transformed cells, in order to (vi) obtain substantially purifiedrecombinant ADAMTS13, e.g. via anion exchange chromatography or affinitychromatography. The term “biologically active derivative” includes alsochimeric molecules such as, e.g. ADAMTS13 (or a biologically activederivative thereof) in combination with Ig, in order to improve thebiological/pharmacological properties such as, e.g. half life ofADAMTS13 in the circulation system of a mammal, particularly human. TheIg could have also the site of binding to an optionally mutated Fcreceptor.

As used herein, the term “thrombus” refers to a blood clot, especially aplatelet-comprising blood clot, a microthrombus, and/or an embolus. Saidthrombus may be attached to an arterial or venous blood vessel or not,and may partially or completely block the blood flow in an arterial orvenous blood vessel.

As used herein, a “therapeutically effective amount or dose” or“sufficient amount or dose” refers to a dose that produces effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

As used herein, a “physiological concentration” of salt refers to a saltconcentration of between about 100 mM and about 200 mM of apharmaceutically acceptable salt. Non-limiting examples ofpharmaceutically acceptable salts include, without limitation, sodiumand potassium chloride, sodium and potassium acetate, sodium andpotassium citrate, sodium and potassium phosphate.

As used herein, a “sub-physiological concentration” of salt refers to asalt concentration of less than about 100 mM of a pharmaceuticallyacceptable salt. In preferred embodiments, a sub-physiologicalconcentration of salt is less than about 80 mM of a pharmaceutical salt.In another preferred embodiment, a sub-physiological concentration ofsalt is less than about 60 mM of a pharmaceutical salt.

As used herein, the term “about” denotes an approximate range of plus orminus 10% from a specified value. For instance, the language “about 20%”encompasses a range of 18-22%. As used herein, about also includes theexact amount. Hence “about 20%” means “about 20%” and also “20%.”

III. ADAMTS13 Compositions and Formulation

In one aspect, the present invention provides stabilized formulations ofplasma derived ADAMTS13 and recombinant ADAMTS13 (rADAMTS13) proteins asdescribed in U.S. Patent Application Publication No. 2011/0229455. Inother embodiments, the formulations provided herein retain significantADAMTS13 activity when stored for extended periods of time. In yet otherembodiments, the formulations of the invention reduce or retarddimerization, oligomerization, and/or aggregation of an ADAMTS13protein.

In one embodiment, the present invention provides formulations ofADAMTS13 comprising a therapeutically effective amount or dose of anADAMTS13 protein, a sub-physiological to physiological concentration ofa pharmaceutically acceptable salt, a stabilizing concentration of oneor more sugars and/or sugar alcohols, a non-ionic surfactant, abuffering agent providing a neutral pH to the formulation, andoptionally a calcium and/or zinc salt. Generally, the stabilizedADAMTS13 formulations provided herein are suitable for pharmaceuticaladministration. In a preferred embodiment, the ADAMTS13 protein is humanADAMTS13 or a biologically active derivative or fragment thereof asdescribed in U.S. Patent Application Publication No. 2011/0229455.

In certain embodiments, the ADAMTS13 formulations are liquidformulations. In other embodiments, the ADAMTS13 formulations arelyophilized formulations that are lyophilized from a liquid formulationas described in U.S. Patent Application Publication No. 2011/0229455. Incertain embodiments of the formulations provided herein, the ADAMTS13protein is a human ADAMTS13 or recombinant human ADAMTS13, or abiologically active derivative or fragment thereof as described in U.S.Patent Application Publication No. 2011/0229455.

In certain embodiments, ADAMTS13 is provided in a therapeuticallyeffective dose between about 0.05 mg/mL and about 10 mg/mL. In otherembodiments, ADAMTS13 is present at a concentration of between about 0.1mg/mL and about 10 mg/mL. In yet other embodiments, ADAMTS13 is presentat a concentration of between about 0.1 mg/mL and about 5 mg/mL. Inanother embodiment, ADAMTS13 is present at a concentration of betweenabout 0.1 mg/mL and about 2 mg/mL. In yet other embodiments, ADAMTS13may be present at about 0.01 mg/mL, or at about 0.02 mg/mL, 0.03 mg/mL,0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL,0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL,1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2.0mg/mL, 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL, 4.0 mg/mL, 4.5 mg/mL, 5.0 mg/mL,5.5 mg/mL, 6.0 mg/mL, 6.5 mg/mL, 7.0 mg/mL, 7.5 mg/mL, 8.0 mg/mL, 8.5mg/mL, 9.0 mg/mL, 9.5 mg/mL, 10.0 mg/mL, or a higher concentration. Inone embodiment, the concentration of a relatively pure ADAMTS13formulation may be determined by spectroscopy (i.e., total proteinmeasured at A280) or other bulk determination (e.g., Bradford assay,silver stain, weight of a lyophilized powder, etc.). In otherembodiments, the concentration of ADAMTS13 may be determined by anADAMTS13 ELISA assay (e.g., mg/mL antigen).

In yet other embodiments, the concentration of ADAMTS13 in a formulationprovided by the present invention may be expressed as a level ofenzymatic activity. For example, in one embodiment an ADAMTS13formulation may contain between about 10 units of FRETS-VWF73 activityand about 10,000 units of FRETS-VWF73 activity or other suitableADAMTS13 enzymatic unit (IU). In other embodiments, the formulation maycontain between about 20 units of FRETS-VWF73 (U_(FV73)) activity andabout 8,000 units of FRETS-VWF73 activity, or between about 30 U_(FV73)and about 6,000 U_(FV73), or between about 40 U_(FV73) and about 4,000U_(FV73), or between about 50 U_(FV73) and about 3,000 U_(FV73), orbetween about 75 U_(FV73) and about 2,500 U_(FV73), or between about 100U_(FV73) and about 2,000 U_(FV73), or between about 200 U_(FV73) andabout 1,500 U_(FV73), or between about other ranges therein. In apreferred embodiment, an ADAMTS13 formulation provided herein containsbetween about 20 and about 10,000. U_(FV73). In certain embodiments, aformulation contains about 10 units of FRETS-VWF73 activity, or about20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500,2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500,3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500,4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500,5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500,6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500,7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500,8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500,9,600, 9,700, 9,800, 9,900, 10,000 or more units of FRETS-VWF73activity.

Similarly, in certain embodiments, the concentration of ADAMTS13 may beexpressed as an enzymatic activity per unit volume, for example,ADAMTS13 enzymatic units per mL (IU/mL). For example, in one embodimentan ADAMTS13 formulation may contain between about 10 IU/mL and about10,000 IU/mL. In other embodiments, the formulation may contain betweenabout 20 IU/mL and about 10,000 IU/mL, or between about 20 IU/mL andabout 8,000 IU/mL, or between about 30 IU/mL and about 6,000 IU/mL, orbetween about 40 IU/mL and about 4,000 IU/mL, or between about 50 IU/mLand about 3,000 IU/mL, or between about 75 IU/mL and about 2,500 IU/mL,or between about 100 IU/mL and about 2,000 IU/mL, or between about 200IU/mL and about 1,500 IU/mL, or between about other ranges therein. In apreferred embodiment, an ADAMTS13 formulation provided herein containsbetween about 150 IU/mL and about 600 IU/mL. In another preferredembodiment, an ADAMTS13 formulation provided herein contains betweenabout 100 IU/mL and about 1,000 IU/mL. In certain embodiments, aformulation contains about 10 IU/mL, or about 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900,2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900,3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900,4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900,5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900,6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900,7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900,8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900,9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900,10,000 or more IU/mL.

In some embodiments, the ADAMTS13 formulations provided herein mayfurther comprise one or more pharmaceutically acceptable excipients,carriers, and/or diluents as described in U.S. Patent Application No.20110229455. Furthermore, in one embodiment, the ADAMTS13 formulationsprovided herein will have a tonicity in a range described in asdescribed in U.S. Patent Application Publication No. 2011/0229455.

In further embodiments, the present invention provides formulations ofADAMTS13 comprising the exemplary formulations described in Section III(“ADAMTS13 Compositions and Formulations”) of U.S. Patent ApplicationPublication No. 2011/0229455.

In certain embodiments ADAMTS13 formulations are produced and comprisethe additives. The methods of ADAMTS13 production and compositionsthereof as described in U.S. Patent Application Publication No.2011/0229455, Sections IV and V, are incorporated herein by reference.

IV. Methods of Treatment

The formulations described herein can be subcutaneously administered fortherapeutic or prophylactic treatments. Generally, for therapeuticapplications, formulations are administered to a subject with a diseaseor condition associated with ADAMTS13 or VWF dysfunction or otherwise inneed thereof, in a “therapeutically effective dose.” Formulations andamounts effective for these uses will depend upon the severity of thedisease or condition and the general state of the patient's health.Single or multiple administrations of the formulations may beadministered depending on the dosage and frequency as required andtolerated by the patient.

A “patient” or “subject” for the purposes of the present inventionincludes both humans and other animals, particularly mammals. Thus thecompositions, formulations, and methods are applicable to both humantherapy and veterinary applications. In a particular embodiment thepatient is a mammal, and in one embodiment, is a human. Other knowntreatments and therapies for conditions associated with ADAMTS13 or VWFdysfunction can be used in combination with the formulations and methodsprovided by the invention.

In certain embodiments, the subcutaneous ADAMTS13 formulation isadministered by subcutaneous injection. In specific embodiments, thesubcutaneous ADAMTS13 formulation is subcutaneously injected into thesame site of a patient (e.g., administered to the upper arm, anteriorsurface of the thigh, lower portion of the abdomen, or upper back) forrepeat or continuous injections. In other embodiments, the subcutaneousADAMTS13 formulation is subcutaneously injected into the different orrotating sites of a patient. In certain embodiments, the subcutaneousADAMTS13 formulation is administered by subcutaneously implanted device.In certain embodiments, the implanted device provides a timed release ofan ADAMTS13 formulation. In certain embodiments, the implanted deviceprovides a continuous release of an ADAMTS13 formulation.

In certain embodiments, an ADAMTS13 formulation described herein is usedfor the treatment and prophylaxis of ADAMTS13 and vWF dysfunction. Incertain embodiments, an ADAMTS13 formulation described herein is usedfor the treatment and prophylaxis of thrombotic diseases and conditions.In certain embodiments, an ADAMTS13 formulation described herein is usedfor the treatment and prophylaxis of an infarction.

In one embodiment, ADAMTS13 is administered at a dose of from 20U_(FV73)/kg body weight to 4000 U_(FV73)/kg body weight. In oneembodiment, ADAMTS13 is administered at a dose of from 20 U_(FV73)/kgbody weight to 2000 U_(FV73)/kg body weight. In one embodiment, ADAMTS13is administered at a dose of from 20 U_(FV73)/kg body weight to 1000U_(FV73)/kg body weight. In one embodiment, ADAMTS13 is administered ata dose of from 20 U_(FV73)/kg body weight to 500 U_(FV73)/kg bodyweight. In one embodiment, ADAMTS13 is administered at a dose of from 20U_(FV73)/kg body weight to 200 U_(FV73)/kg body weight. In oneembodiment, ADAMTS13 is administered at a dose of from 20 U_(FV73)/kgbody weight to 100 U_(FV73)/kg body weight. In one embodiment, ADAMTS13is administered at a dose of from 40 U_(FV73)/kg body weight to 200U_(FV73)/kg body weight. In one embodiment, ADAMTS13 is administered ata dose of from 40 U_(FV73)/kg body weight to 100 U_(FV73)/kg bodyweight. In other embodiments, ADAMTS13 is administered at about 20U_(FV73)/kg body weight, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300,2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300,3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000 U_(FV73)/kg body weight,or at an intermediate concentration or concentration range thereof.

Generally, the dose of ADAMTS13 administered to a mammal will dependupon, among other factors, the disease or condition being treated, thespecies of the mammal, the age of the mammal, and the overall health ofthe mammal. The skilled person will readily be able to translate dosagesbetween different mammals, for example from a mouse dose to a humandose. One means for extrapolating a human dose from an animal doseincludes the use of body surface area, which is known to correlate wellwith several metabolic parameters, e.g., blood volume, circulatingplasma, and renal function, in diverse mammals. Thus, a conversionfactor (e.g., K_(m)) correlating an average weight of a mammal to anaverage body surface area can be used to correlate a drug dosage (e.g.,a dosage of ADAMTS13), expressed in units of protein (e.g., mass oractivity) per body weight of the mammal (e.g., kg), used for a firsttype of mammal (e.g., a mouse) with a corresponding dose in a secondtype of mammal (e.g., a human). For review, see Reagan-Shaw et al.,FASEB, 22:659-62 (2007). For example, this can be done by firstmultiplying the drug dosage administered to the first type of mammal bythe conversion factor determined for that mammal, and then dividing theproduct by the conversion factor determined for the second type ofmammal. Examples of such conversion factors are given below in Table 1,adopted from the U.S. Department of Health and Human Services guidelinesfor estimating the maximum safe starting dose in initial clinical trialsfor therapeutics in adult healthy volunteers.

TABLE 1 Conversion of animal doses to human equivalent doses based onbody surface area. To Convert Animal Dose in To Convert Animal Dose inmg/kg mg/kg to Dose in to HED^(a) in mg/kg, Either: mg/m², MultiplyDivide Multiply Species by k_(m) Animal Dose By Animal Dose By Human 37— — Child (20 kg)^(b) 25 — — Mouse 3 12.3 0.08 Hamster 5 7.4 0.13 Rat 66.2 0.16 Ferret 7 5.3 0.19 Guinea pig 8 4.6 0.22 Rabbit 12 3.1 0.32 Dog20 1.8 0.54 Primates: Monkeys^(c) 12 3.1 0.32 Marmoset 6 6.2 0.16Squirrel 7 5.3 0.19 monkey Baboon 20 1.8 0.54 Micro-pig 27 1.4 0.73Mini-pig 35 1.1 0.95 ^(a)Assumes 60 kg human. For species not listed orfor weights outside the sandard ranges. HED can be calculated from thefollowing formula: HED = animal dose in mg/kg × (animal weight inkg/human weight in kg)^(0.33). ^(b)This k_(m) value is provided forreference only since healthy children will rarely be volunteers forphase 1 trials. ^(c)For example, cynomolgus, rhesus, and stumptail.

In certain embodiments, the bioavailability of the ADAMTS13 aftersubcutaneous administration is between at least 50% and at least 80% ascompared to intravenous administration normalized for the same dose. Incertain embodiments, the bioavailability of the ADAMTS13 aftersubcutaneous administration is between at least 60% and at least 80% ascompared to intravenous administration normalized for the same dose. Incertain embodiments, the bioavailability of the ADAMTS13 aftersubcutaneous administration is between at least 50% and 70% as comparedto intravenous administration normalized for the same dose. In certainembodiments, the bioavailability of the ADAMTS13 after subcutaneousadministration is between at least 55% and 65% as compared tointravenous administration normalized for the same dose. In certainembodiments, the bioavailability of the ADAMTS13 after subcutaneousadministration is between at least 55% and 70% as compared tointravenous administration normalized for the same dose.

In certain embodiments, the bioavailability of the ADAMTS13 aftersubcutaneous administration is at least 40%, or at least 45%, or atleast 50%, or at least 51%, or at least 52%, or at least 53%, or atleast 54%, or at least 55%, or at least 56%, or at least 57%, or atleast 58%, or at least 59%, or at least 60%, or at least 61%, or atleast 62%, or at least 63%, or at least 64%, or at least 65%, or atleast 66%, or at least 67%, or at least 68%, or at least 69%, or atleast 70%, or at least 71%, or at least 72%, or at least 73%, or atleast 74%, or at least 75%, or at least 76%, or at least 77%, or atleast 78%, or at least 79%, or at least 80%, or at least 81%, or atleast 82%, or at least 83%, or at least 84%, or at least 85% as comparedto intravenous administration normalized for the same dose.

In certain embodiments, the ADAMTS13 formulation is subcutaneouslyadministered in a single bolus injection. In certain embodiments, theADAMTS13 formulation is subcutaneously administered monthly. In certainembodiments, the ADAMTS13 formulation is subcutaneously administeredevery two weeks. In certain embodiments, the ADAMTS13 formulation issubcutaneously administered weekly. In certain embodiments, the ADAMTS13formulation is subcutaneously administered twice a week. In certainembodiments, the ADAMTS13 formulation is subcutaneously administereddaily. In certain embodiments, the ADAMTS13 formulation issubcutaneously administered every 12 hours. In certain embodiments, theADAMTS13 formulation is subcutaneously administered every 8 hours. Incertain embodiments, the ADAMTS13 formulation is subcutaneouslyadministered every six hours. In certain embodiments, the ADAMTS13formulation is subcutaneously administered every four hours. In certainembodiments, the ADAMTS13 formulation is subcutaneously administeredevery two hours.

In one embodiment, the ADAMTS13 formulation is subcutaneouslyadministered in a dose and frequency combination selected fromvariations 1 to 1133 in Table 2. Generally, the dose and frequency ofADAMTS13 administered to a mammal will depend upon, among other factors,the disease or condition being treated, the species of the mammal, theage of the mammal, and the overall health of the mammal.

TABLE 2 Useful combinations of ADAMTS13 dosage and frequency forsubcutaneous administration. Dose One Two Three One Two Every One TwoThree Four Six (UFV73/kg Time Times Times time Times Other Time TimesTimes Times Times body weight) Monthly Monthly Monthly Weekly Weekly DayDaily Daily Daily Daily Daily  20-4000 Var. 1 Var. 104 Var. 207 Var. 310Var. 413 Var. Var. Var. Var. Var. Var. 516 619 722 825 928 1031  20-3000Var. 2 Var. 105 Var. 208 Var. 311 Var. 414 Var. Var. Var. Var. Var. Var.517 620 723 826 929 1032  20-2000 Var. 3 Var. 106 Var. 209 Var. 312 Var.415 Var. Var. Var. Var. Var. Var. 518 621 724 827 930 1033  20-1000 Var.4 Var. 107 Var. 210 Var. 313 Var. 416 Var. Var. Var. Var. Var. Var. 519622 725 828 931 1034  20-750 Var. 5 Var. 108 Var. 211 Var. 314 Var. 417Var. Var. Var. Var. Var. Var. 520 623 726 829 932 1035  20-500 Var. 6Var. 109 Var. 212 Var. 315 Var. 418 Var. Var. Var. Var. Var. Var. 521624 727 830 933 1036  20-250 Var. 7 Var. 110 Var. 213 Var. 316 Var. 419Var. Var. Var. Var. Var. Var. 522 625 728 831 934 1037  20-200 Var. 8Var. 111 Var. 214 Var. 317 Var. 420 Var. Var. Var. Var. Var. Var. 523626 729 832 935 1038  20-150 Var. 9 Var. 112 Var. 215 Var. 318 Var. 421Var. Var. Var. Var. Var. Var. 524 627 730 833 936 1039  20-100 Var. 10Var. 113 Var. 216 Var. 319 Var. 422 Var. Var. Var. Var. Var. Var. 525628 731 834 937 1040 20-75 Var. 11 Var. 114 Var. 217 Var. 320 Var. 423Var. Var. Var. Var. Var. Var. 526 629 732 835 938 1041 20-50 Var. 12Var. 115 Var. 218 Var. 321 Var. 424 Var. Var. Var. Var. Var. Var. 527630 733 836 939 1042 20-40 Var. 13 Var. 116 Var. 219 Var. 322 Var. 425Var. Var. Var. Var. Var. Var. 528 631 734 837 940 1043  40-4000 Var. 14Var. 117 Var. 220 Var. 323 Var. 426 Var. Var. Var. Var. Var. Var. 529632 735 838 941 1044  40-3000 Var. 15 Var. 118 Var. 221 Var. 324 Var.427 Var. Var. Var. Var. Var. Var. 530 633 736 839 942 1045  40-2000 Var.16 Var. 119 Var. 222 Var. 325 Var. 428 Var. Var. Var. Var. Var. Var. 531634 737 840 943 1046  40-1000 Var. 17 Var. 120 Var. 223 Var. 326 Var.429 Var. Var. Var. Var. Var. Var. 532 635 738 841 944 1047  40-750 Var.18 Var. 121 Var. 224 Var. 327 Var. 430 Var. Var. Var. Var. Var. Var. 533636 739 842 945 1048  40-500 Var. 19 Var. 122 Var. 225 Var. 328 Var. 431Var. Var. Var. Var. Var. Var. 534 637 740 843 946 1049  40-250 Var. 20Var. 123 Var. 226 Var. 329 Var. 432 Var. Var. Var. Var. Var. Var. 535638 741 844 947 1050  40-200 Var. 21 Var. 124 Var. 227 Var. 330 Var. 433Var. Var. Var. Var. Var. Var. 536 639 742 845 948 1051  40-150 Var. 22Var. 125 Var. 228 Var. 331 Var. 434 Var. Var. Var. Var. Var. Var. 537640 743 846 949 1052  40-100 Var. 23 Var. 126 Var. 229 Var. 332 Var. 435Var. Var. Var. Var. Var. Var. 538 641 744 847 950 1053 40-75 Var. 24Var. 127 Var. 230 Var. 333 Var. 436 Var. Var. Var. Var. Var. Var. 539642 745 848 951 1054  60-4000 Var. 25 Var. 128 Var. 231 Var. 334 Var.437 Var. Var. Var. Var. Var. Var. 540 643 746 849 952 1055  60-3000 Var.26 Var. 129 Var. 232 Var. 335 Var. 438 Var. Var. Var. Var. Var. Var. 541644 747 850 953 1056  60-2000 Var. 27 Var. 130 Var. 233 Var. 336 Var.439 Var. Var. Var. Var. Var. Var. 542 645 748 851 954 1057  60-1000 Var.28 Var. 131 Var. 234 Var. 337 Var. 440 Var. Var. Var. Var. Var. Var. 543646 749 852 955 1058  60-750 Var. 29 Var. 132 Var. 235 Var. 338 Var. 441Var. Var. Var. Var. Var. Var. 544 647 750 853 956 1059  60-500 Var. 30Var. 133 Var. 236 Var. 339 Var. 442 Var. Var. Var. Var. Var. Var. 545648 751 854 957 1060  60-250 Var. 31 Var. 134 Var. 237 Var. 340 Var. 443Var. Var. Var. Var. Var. Var. 546 649 752 855 958 1061  60-200 Var. 32Var. 135 Var. 238 Var. 341 Var. 444 Var. Var. Var. Var. Var. Var. 547650 753 856 959 1062  60-150 Var. 33 Var. 136 Var. 239 Var. 342 Var. 445Var. Var. Var. Var. Var. Var. 548 651 754 857 960 1063  60-100 Var. 34Var. 137 Var. 240 Var. 343 Var. 446 Var. Var. Var. Var. Var. Var. 549652 755 858 961 1064  80-4000 Var. 35 Var. 138 Var. 241 Var. 344 Var.447 Var. Var. Var. Var. Var. Var. 550 653 756 859 962 1065  80-3000 Var.36 Var. 139 Var. 242 Var. 345 Var. 448 Var. Var. Var. Var. Var. Var. 551654 757 860 963 1066  80-2000 Var. 37 Var. 140 Var. 243 Var. 346 Var.449 Var. Var. Var. Var. Var. Var. 552 655 758 861 964 1067  80-1000 Var.38 Var. 141 Var. 244 Var. 347 Var. 450 Var. Var. Var. Var. Var. Var. 553656 759 862 965 1068  80-750 Var. 39 Var. 142 Var. 245 Var. 348 Var. 451Var. Var. Var. Var. Var. Var. 554 657 760 863 966 1069  80-500 Var. 40Var. 143 Var. 246 Var. 349 Var. 452 Var. Var. Var. Var. Var. Var. 555658 761 864 967 1070  80-250 Var. 41 Var. 144 Var. 247 Var. 350 Var. 453Var. Var. Var. Var. Var. Var. 556 659 762 865 968 1071  80-200 Var. 42Var. 145 Var. 248 Var. 351 Var. 454 Var. Var. Var. Var. Var. Var. 557660 763 866 969 1072  80-150 Var. 43 Var. 146 Var. 249 Var. 352 Var. 455Var. Var. Var. Var. Var. Var. 558 661 764 867 970 1073  80-100 Var. 44Var. 147 Var. 250 Var. 353 Var. 456 Var. Var. Var. Var. Var. Var. 559662 765 868 971 1074  100-4000 Var. 45 Var. 148 Var. 251 Var. 354 Var.457 Var. Var. Var. Var. Var. Var. 560 663 766 869 972 1075  100-3000Var. 46 Var. 149 Var. 252 Var. 355 Var. 458 Var. Var. Var. Var. Var.Var. 561 664 767 870 973 1076  100-2000 Var. 47 Var. 150 Var. 253 Var.356 Var. 459 Var. Var. Var. Var. Var. Var. 562 665 768 871 974 1077 100-1000 Var. 48 Var. 151 Var. 254 Var. 357 Var. 460 Var. Var. Var.Var. Var. Var. 563 666 769 872 975 1078 100-750 Var. 49 Var. 152 Var.255 Var. 358 Var. 461 Var. Var. Var. Var. Var. Var. 564 667 770 873 9761079 100-500 Var. 50 Var. 153 Var. 256 Var. 359 Var. 462 Var. Var. Var.Var. Var. Var. 565 668 771 874 977 1080 100-250 Var. 51 Var. 154 Var.257 Var. 360 Var. 463 Var. Var. Var. Var. Var. Var. 566 669 772 875 9781081 100-200 Var. 52 Var. 155 Var. 258 Var. 361 Var. 464 Var. Var. Var.Var. Var. Var. 567 670 773 876 979 1082 100-150 Var. 53 Var. 156 Var.259 Var. 362 Var. 465 Var. Var. Var. Var. Var. Var. 568 671 774 877 9801083  200-4000 Var. 54 Var. 157 Var. 260 Var. 363 Var. 466 Var. Var.Var. Var. Var. Var. 569 672 775 878 981 1084  200-3000 Var. 55 Var. 158Var. 261 Var. 364 Var. 467 Var. Var. Var. Var. Var. Var. 570 673 776 879982 1085  200-2000 Var. 56 Var. 159 Var. 262 Var. 365 Var. 468 Var. Var.Var. Var. Var. Var. 571 674 777 880 983 1086  200-1000 Var. 57 Var. 160Var. 263 Var. 366 Var. 469 Var. Var. Var. Var. Var. Var. 572 675 778 881984 1087 200-750 Var. 58 Var. 161 Var. 264 Var. 367 Var. 470 Var. Var.Var. Var. Var. Var. 573 676 779 882 985 1088 200-500 Var. 59 Var. 162Var. 265 Var. 368 Var. 471 Var. Var. Var. Var. Var. Var. 574 677 780 883986 1089 200-250 Var. 60 Var. 163 Var. 266 Var. 369 Var. 472 Var. Var.Var. Var. Var. Var. 575 678 781 884 987 1090  400-4000 Var. 61 Var. 164Var. 267 Var. 370 Var. 473 Var. Var. Var. Var. Var. Var. 576 679 782 885988 1091  400-3000 Var. 62 Var. 165 Var. 268 Var. 371 Var. 474 Var. Var.Var. Var. Var. Var. 577 680 783 886 989 1092  400-2000 Var. 63 Var. 166Var. 269 Var. 372 Var. 475 Var. Var. Var. Var. Var. Var. 578 681 784 887990 1093  400-1000 Var. 64 Var. 167 Var. 270 Var. 373 Var. 476 Var. Var.Var. Var. Var. Var. 579 682 785 888 991 1094 400-750 Var. 65 Var. 168Var. 271 Var. 374 Var. 477 Var. Var. Var. Var. Var. Var. 580 683 786 889992 1095 400-500 Var. 66 Var. 169 Var. 272 Var. 375 Var. 478 Var. Var.Var. Var. Var. Var. 581 684 787 890 993 1096  600-4000 Var. 67 Var. 170Var. 273 Var. 376 Var. 479 Var. Var. Var. Var. Var. Var. 582 685 788 891994 1097  600-3000 Var. 68 Var. 171 Var. 274 Var. 377 Var. 480 Var. Var.Var. Var. Var. Var. 583 686 789 892 995 1098  600-2000 Var. 69 Var. 172Var. 275 Var. 378 Var. 481 Var. Var. Var. Var. Var. Var. 584 687 790 893996 1099  600-1000 Var. 70 Var. 173 Var. 276 Var. 379 Var. 482 Var. Var.Var. Var. Var. Var. 585 688 791 894 997 1100 600-750 Var. 71 Var. 174Var. 277 Var. 380 Var. 483 Var. Var. Var. Var. Var. Var. 586 689 792 895998 1101 1000-4000 Var. 72 Var. 175 Var. 278 Var. 381 Var. 484 Var. Var.Var. Var. Var. Var. 587 690 793 896 999 1102 1000-3000 Var. 73 Var. 176Var. 279 Var. 382 Var. 485 Var. Var. Var. Var. Var. Var. 588 691 794 8971000 1103 1000-2000 Var. 74 Var. 177 Var. 280 Var. 383 Var. 486 Var.Var. Var. Var. Var. Var. 589 692 795 898 1001 1104 2000-4000 Var. 75Var. 178 Var. 281 Var. 384 Var. 487 Var. Var. Var. Var. Var. Var. 590693 796 899 1002 1105 2000-3000 Var. 76 Var. 179 Var. 282 Var. 385 Var.488 Var. Var. Var. Var. Var. Var. 591 694 797 900 1003 1106 3000-4000Var. 77 Var. 180 Var. 283 Var. 386 Var. 489 Var. Var. Var. Var. Var.Var. 592 695 798 901 1004 1107  20 ± 10% Var. 78 Var. 181 Var. 284 Var.387 Var. 490 Var. Var. Var. Var. Var. Var. 593 696 799 902 1005 1108  40± 10% Var. 79 Var. 182 Var. 285 Var. 388 Var. 491 Var. Var. Var. Var.Var. Var. 594 697 800 903 1006 1109  60 ± 10% Var. 80 Var. 183 Var. 286Var. 389 Var. 492 Var. Var. Var. Var. Var. Var. 595 698 801 904 10071110  80 ± 10% Var. 81 Var. 184 Var. 287 Var. 390 Var. 493 Var. Var.Var. Var. Var. Var. 596 699 802 905 1008 1111 100 ± 10% Var. 82 Var. 185Var. 288 Var. 391 Var. 494 Var. Var. Var. Var. Var. Var. 597 700 803 9061009 1112 150 ± 10% Var. 83 Var. 186 Var. 289 Var. 392 Var. 495 Var.Var. Var. Var. Var. Var. 598 701 804 907 1010 1113 200 ± 10% Var. 84Var. 187 Var. 290 Var. 393 Var. 496 Var. Var. Var. Var. Var. Var. 599702 805 908 1011 1114 250 ± 10% Var. 85 Var. 188 Var. 291 Var. 394 Var.497 Var. Var. Var. Var. Var. Var. 600 703 806 909 1012 1115 300 ± 10%Var. 86 Var. 189 Var. 292 Var. 395 Var. 498 Var. Var. Var. Var. Var.Var. 601 704 807 910 1013 1116 350 ± 10% Var. 87 Var. 190 Var. 293 Var.396 Var. 499 Var. Var. Var. Var. Var. Var. 602 705 808 911 1014 1117 400± 10% Var. 88 Var. 191 Var. 294 Var. 397 Var. 500 Var. Var. Var. Var.Var. Var. 603 706 809 912 1015 1118 450 ± 10% Var. 89 Var. 192 Var. 295Var. 398 Var. 501 Var. Var. Var. Var. Var. Var. 604 707 810 913 10161119 500 ± 10% Var. 90 Var. 193 Var. 296 Var. 399 Var. 502 Var. Var.Var. Var. Var. Var. 605 708 811 914 1017 1120 600 ± 10% Var. 91 Var. 194Var. 297 Var. 400 Var. 503 Var. Var. Var. Var. Var. Var. 606 709 812 9151018 1121 700 ± 10% Var. 92 Var. 195 Var. 298 Var. 401 Var. 504 Var.Var. Var. Var. Var. Var. 607 710 813 916 1019 1122 800 ± 10% Var. 93Var. 196 Var. 299 Var. 402 Var. 505 Var. Var. Var. Var. Var. Var. 608711 814 917 1020 1123 900 ± 10% Var. 94 Var. 197 Var. 300 Var. 403 Var.506 Var. Var. Var. Var. Var. Var. 609 712 815 918 1021 1124 1000 ± 10% Var. 95 Var. 198 Var. 301 Var. 404 Var. 507 Var. Var. Var. Var. Var.Var. 610 713 816 919 1022 1125 1250 ± 10%  Var. 96 Var. 199 Var. 302Var. 405 Var. 508 Var. Var. Var. Var. Var. Var. 611 714 817 920 10231126 1500 ± 10%  Var. 97 Var. 200 Var. 303 Var. 406 Var. 509 Var. Var.Var. Var. Var. Var. 612 715 818 921 1024 1127 1750 ± 10%  Var. 98 Var.201 Var. 304 Var. 407 Var. 510 Var. Var. Var. Var. Var. Var. 613 716 819922 1025 1128 2000 ± 10%  Var. 99 Var. 202 Var. 305 Var. 408 Var. 511Var. Var. Var. Var. Var. Var. 614 717 820 923 1026 1129 2500 ± 10%  Var.Var. 203 Var. 306 Var. 409 Var. 512 Var. Var. Var. Var. Var. Var. 100615 718 821 924 1027 1130 3000 ± 10%  Var. Var. 204 Var. 307 Var. 410Var. 513 Var. Var. Var. Var. Var. Var. 101 616 719 822 925 1028 11313500 ± 10%  Var. Var. 205 Var. 308 Var. 411 Var. 514 Var. Var. Var. Var.Var. Var. 102 617 720 823 926 1029 1132 4000 ± 10%  Var. Var. 206 Var.309 Var. 412 Var. 515 Var. Var. Var. Var. Var. Var. 103 618 721 824 9271030 1133

In certain embodiments, about 120-300% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 120% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 130% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 140% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 150% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 160% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 170% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 180% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 190% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.

In a specific embodiment, about 200% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 210% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 220% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 230% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 240% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 250% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 260% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 270% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 280% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 290% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.In a specific embodiment, about 300% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.

In certain embodiments, about 120%, about 122%, about 125%, about 127%,about 130%, about 132%, about 135%, about 137%, about 140%, about 142%,about 145%, about 147%, about 150%, about 152%, about 155%, about 157%,about 160%, about 162%, about 165%, about 167%, about 170%, about 172%,about 175%, about 177%, about 180%, about 182%, about 185%, about 187%,about 190%, about 192%, about 195%, about 97%, about 200%, about 202%,about 205%, about 207%, about 210%, about 212%, about 215%, about 217%,about 220%, about 222%, about 225%, about 227%, about 230%, about 232%,about 235%, about 237%, about 240%, about 242%, about 245%, about 247%,about 250%, about 252%, about 255%, about 257%, about 260%, about 262%,about 265%, about 267%, about 270%, about 272%, about 275%, about 277%,about 280%, about 282%, about 285%, about 287%, about 290%, about 292%,about 295%, about 297%, or about 300% of the amount of a standardintravenous dose of an ADAMTS13 formulation for a specific indication asmeasured in activity units per kilogram is administered subcutaneously.

In one embodiment, an ADAMTS13 formulation is administeredsubcutaneously to reduce inflammation caused by the clotting disorder(e.g., an infarction), thereby preventing or reducing tissue damage(e.g., damage to the cerebral damage) and/or to reduce reperfusioninjury by preventing leukocyte infiltration and damage. In oneembodiment, an ADAMTS13 formulation is administered subcutaneously toprotect against secondary injury to infarct tissue (e.g., cerebraltissue and myocardial tissue) caused by reperfusion.

Inherited TTP

In one embodiment, an ADAMTS13 formulation described herein is used forthe treatment and prophylaxis of inherited TTP Inherited TTP is due togenetic mutations of the ADAMTS13 gene. Inherited TTP can lead toneurologic manifestations (e.g., mental status, stroke, seizures,hemiplegia, paresthesias, visual disturbance, and aphasia), fatigue, andsevere bleeding. If left untreated, acquired TTP can be fatal or cancause lasting physiological damage. Furthermore, because inherited TTPis due to a genetic mutation, life-long treatment is needed and patientcompliance is required. While intravenous delivery of ADAMTS13formulations is effective at treating inherited TTP, intravenousdelivery of drugs is not easy for patients to handle (especiallychildren with inherited TTP) and decreases patient compliance.Accordingly, it would be beneficial to develop a subcutaneous ADAMTS13formulation and a method of ADAMTS13 subcutaneous delivery.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 4000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 4000 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 4000 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 4000 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to4000 U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. Inone embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredabout once every 12 hours.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 2000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 2000 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 2000 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 2000 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to2000 U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. Inone embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredabout once every 12 hours.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 1000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 1000 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 1000 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 1000 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to1000 U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. Inone embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredabout once every 12 hours.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 500 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 500 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 500 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 500 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to 500U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 200 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 200 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to 200U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 100 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 100 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 100 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 100 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to 100U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 40 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 40 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 40 to 200 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 40to 200 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 40 to 200U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatinginherited TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 and frequency of the dosingis selected from variations 1 to 1133 in Table 2. In a specificembodiment, the mammal is a human.

Acquired TTP

In specific embodiments, an ADAMTS13 formulation described herein isused for the treatment and prophylaxis of acquired TTP. In acquired TTP,patients have a low ADAMTS13 activity due to the development ofautoimmune antibodies directed at ADAMTS13. Immune-complexed ADAMTS13 isinactivated, neutralized and/or cleared from the blood stream andpatient plasma. Reduced ADAMTS13 activity leads to the accumulation oflarge uncleaved VWF multimers which can spontaneously adhere toplatelets and leading to platelet-VWF-rich thrombi in themicrocirculation. Like inherited TTP, acquired TTP can also lead toneurologic manifestations (e.g., mental status, stroke, seizures,hemiplegia, paresthesias, visual disturbance, and aphasia), fatigue, andsevere bleeding. If left untreated, acquired TTP can be fatal or cancause lasting physiological damage. Accordingly, patient compliance ofADAMTS13 administration is necessary to prevent permanent damage andeventual fatalities. Thus, it would be beneficial to develop asubcutaneous ADAMTS13 formulation and a method of ADAMTS13 subcutaneousdelivery to increase patient ease and compliance as described above.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 4000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 4000 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 4000 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 4000 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to4000 U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. Inone embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredabout once every 12 hours.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 2000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 2000 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 2000 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 2000 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to2000 U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. Inone embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredabout once every 12 hours.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 1000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 1000 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 1000 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 1000 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to1000 U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. Inone embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredabout once every 12 hours.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 500 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 500 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 500 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 500 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to 500U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 200 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 200 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to 200U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 100 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 100 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 100 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 20to 100 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 20 to 100U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 40 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 40 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 40 to 200 U_(FV73)/kgADAMTS13 is administered about twice a month. In one embodiment, the 40to 200 U_(FV73)/kg ADAMTS13 is administered about once a week. In oneembodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administered abouttwice a week. In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 isadministered about once every 48 hours. In one embodiment, the 40 to 200U_(FV73)/kg ADAMTS13 is administered about once every 24 hours. In oneembodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administered aboutonce every 12 hours.

In one embodiment, the disclosure provides a method for treatingacquired TTP in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 and frequency of the dosingis selected from variations 1 to 1133 in Table 2. In a specificembodiment, the mammal is a human.

Myocardial Infarction and Reperfusion Injury

In specific embodiments, an ADAMTS13 formulation described herein isused for the treatment and prophylaxis of myocardial infarction. Inspecific embodiments, an ADAMTS13 formulation described herein is usedfor the treatment and prophylaxis of ischemic/reperfusion injury.Reperfusion is the restoration of blood supply to tissue that isischemic, due to decrease in blood supply. Reperfusion is a procedurefor treating infarction (e.g., myocardial infarction and cerebralinfarction) or other ischemia, by enabling viable ischemic tissue torecover, thus limiting further necrosis. However, reperfusion can itselffurther damage the ischemic tissue, causing reperfusion injury. Forexample, acute myocardial infarction (AMI) is caused by thromboticocclusion of a coronary artery. In addition to the immediate injury thatoccurs during deprivation of blood flow, ischemic/reperfusion injuryinvolves tissue injury that occurs after blood flow is restored from thereperfusion.

Furthermore, it has been reported that ADAMTS13 has an anti-inflammatoryeffect that prevents or decreases secondary injury during ischemicreperfusion. De Meyer et al. (“Protective anti-inflammatory effect ofADAMTS13 on myocardial ischemia/reperfusion injury in mice,” Blood,2012, 120(26):5217-5223). As described by De Meyer et al., VWF andADAMTS13 are involved in platelet adhesion and thrombus formationbecause ADAMTS13 cleaves the most thrombogenic VWF multimers intosmaller and less hemostatically active VWF fragments. De Meyer et al.also describe ADAMTS's role in down-regulating inflammatory responses.It has also been shown that ADAMTS13 can reduce thrombosis andinflammation (e.g., atherosclerosis). Chauhan et al. (“ADAMTS13: a newlink between thrombosis and inflammation,” J Exp Med., 2008,205:2065-2074); Chauhan et al. (“Systemic antithrombotic effects ofADAMTS13,” J Exp Med., 2006, 203:767-776; Gandhi et al. (“ADAMTS13reduces vascular inflammation and the development of earlyatherosclerosis in mice,” Blood, 2012, 119(10):2385-2391.

De Meyer et al. suggest that ADAMTS13 prevents excessive VWF-mediatedplatelet and leukocyte recruitment in the ischemic myocardium bycleaving VWF. Based on this hypothesis, De Meyer et al. show thatneutrophil infiltration in the myocardium of animals with inducedmyocardial infarction was nine times lower when the animals were treatedADAMTS13. Accordingly, De Meyer et al. show that ADAMTS13 reducesinflammatory responses in ischemic myocardium. This reduced inflammationalso reduces reperfusion injury by preventing leukocyte infiltration anddamage. Thus, it would be beneficial to subcutaneously administer anADAMTS13 formulation to patients to avoid inflammation that results intissue damage during infarction (e.g., myocardial infarction andcerebral infaction) and reperfusion because subcutaneous administrationis easier and faster to administer than intravenous compositions thatare generally administered by a medical professional.

In some embodiments, the pharmaceutical composition is administeredimmediately upon discovery of a myocardial infarction, e.g., within 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90minutes, 110 minutes, 120 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, 24 hours, 25 or more hours, or anycombination thereof, for treatment of the infarction and/or reperfusioninjury. Accordingly, it is important to have a pharmaceuticalcomposition that can be quickly and easily administered.

In one embodiment, the disclosure provides a method for treatingmyocardial infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 4000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a myocardial infarction in the mammal. Inone embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a myocardial infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingmyocardial infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 2000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a myocardial infarction in the mammal. Inone embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a myocardial infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingmyocardial infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 1000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a myocardial infarction in the mammal. Inone embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a myocardial infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingmyocardial infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 500 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a myocardial infarction in the mammal. Inone embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a myocardial infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingmyocardial infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a myocardial infarction in the mammal. Inone embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a myocardial infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingmyocardial infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 100 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a myocardial infarction in the mammal. Inone embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a myocardial infarction in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a myocardial infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingmyocardial infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 40 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 40 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a myocardial infarction in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a myocardial infarction in the mammal. Inone embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a myocardial infarction in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a myocardial infarction in the mammal.

Cerebral Infarction

In one embodiment, an ADAMTS13 formulation described herein is used forthe treatment and/or prophylaxis of cerebral infarction. Cerebralinfarction, commonly referred to as a stroke, occurs when blood flow topart of the brain is prevented. Cerebral infarctions can occur, forexample, when a blood vessel that supplies blood to the brain is blockedby a blood clot. A Cerebral infarction can also be the result of a bluntforce trauma and mechanical injury. This can either be caused by a clotin an artery of the brain (thrombotic stroke) or by a clot from anotherpart of the body that travels to the brain (embolic stroke).Accordingly, in some embodiments, the invention provides a method ofimproving the recovery of (or reducing the damage to) sensory and/ormotor function in a patient after a cerebral infarction, comprising thestep of administering to the individual a pharmaceutical compositioncomprising a therapeutically effective amount of an ADAMTS13 protein ora biologically active derivative thereof, thereby improving the recoveryof (or reducing the damage to) sensory and/or motor function in theindividual post-cerebral infarction.

In some embodiments, the pharmaceutical composition is administeredimmediately upon discovery of a cerebral infarction, e.g., within 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90minutes, 110 minutes, 120 minutes, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21hours, 22 hours, 23 hours, 24 hours, 25 or more hours, or anycombination thereof. Accordingly, it is important to have apharmaceutical composition that can be quickly and easily administered.

In one embodiment, the disclosure provides a method for treatingcerebral infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 4000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a cerebral infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingcerebral infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 2000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a cerebral infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingcerebral infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 1000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a cerebral infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingcerebral infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 500 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a cerebral infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingcerebral infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a cerebral infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingcerebral infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 100 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a cerebral infarction in the mammal.

In one embodiment, the disclosure provides a method for treatingcerebral infarction in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 40 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 40 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a cerebral infarction in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a cerebral infarction in the mammal. Inone embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a cerebral infarction in the mammal.

Deep Vein Thrombosis

In one embodiment, an ADAMTS13 formulation described herein is used forthe treatment and/or prophylaxis of deep vein thrombosis (DVT). DVT is ablood clot that forms in a vein, deep in the body. While most deep veinclots occur in the lower leg or thigh, they can occur throughout thebody. DVT is a particularly dangerous disease because a blood clot canbreak off and travel through the bloodstream (an embolus) to the heart,lungs, or brain, for example. Such embolisms can cause damage to organsand may result in death. Accordingly, as described above, ADAMTS13formulations can be used to treat DVT and resulting embolisms.Furthermore, because DVT can develop and cause damage quickly, it isimportant to have a pharmaceutical composition that can be quickly andeasily administered. Thus, it would be beneficial to develop asubcutaneous ADAMTS13 formulation and a method of ADAMTS13 subcutaneousdelivery.

In some embodiments, an ADAMTS13 pharmaceutical composition isadministered immediately upon discovery of a deep vein thrombosis, e.g.,within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60minutes, 90 minutes, 110 minutes, 120 minutes, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 or more hours, or anycombination thereof. Accordingly, it is important to have apharmaceutical composition that can be quickly and easily administered.

In one embodiment, the disclosure provides a method for treating deepvein thrombosis in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 4000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a deep vein thrombosis in the mammal.

In one embodiment, the disclosure provides a method for treating deepvein thrombosis in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 2000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a deep vein thrombosis in the mammal.

In one embodiment, the disclosure provides a method for treating deepvein thrombosis in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 1000 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a deep vein thrombosis in the mammal.

In one embodiment, the disclosure provides a method for treating deepvein thrombosis in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 500 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a deep vein thrombosis in the mammal.

In one embodiment, the disclosure provides a method for treating deepvein thrombosis in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a deep vein thrombosis in the mammal.

In one embodiment, the disclosure provides a method for treating deepvein thrombosis in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 20 to 100 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 20 U_(FV73) to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a deep vein thrombosis in the mammal.

In one embodiment, the disclosure provides a method for treating deepvein thrombosis in a mammal in need thereof, the method includingsubcutaneously administering a therapeutically effective amount of acomposition comprising isolated ADAMTS13 to the mammal, where thetherapeutically effective amount of ADAMTS13 is from 40 to 200 units ofFRETS-VWF73 activity per kilogram body weight of the mammal(U_(FV73)/kg). In a specific embodiment, the mammal is a human. In oneembodiment, the 40 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 10 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 30 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 60 minutes of discovery of a deep vein thrombosis in the mammal.In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 4 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 12 hours of discovery of a deep vein thrombosis in the mammal. Inone embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administeredwithin 24 hours of discovery of a deep vein thrombosis in the mammal.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal in need therein, where the therapeutically effective amount ofADAMTS13 is from 20 to 4000 units of FRETS-VWF73 activity per kilogrambody weight of the mammal (UFV73/kg). In a specific embodiment, themammal is a human. In one embodiment, the 20 UFV73 to 4000 UFV73/kgADAMTS13 is administered about once a month. In one embodiment, the 20to 4000 UFV73/kg ADAMTS13 is administered about twice a month. In oneembodiment, the 20 to 4000 UFV73/kg ADAMTS13 is administered about oncea week. In one embodiment, the 20 to 4000 UFV73/kg ADAMTS13 isadministered about twice a week. In one embodiment, the 20 to 4000UFV73/kg ADAMTS13 is administered about once every 48 hours. In oneembodiment, the 20 to 4000 UFV73/kg ADAMTS13 is administered about onceevery 24 hours. In one embodiment, the 20 to 4000 UFV73/kg ADAMTS13 isadministered about once every 12 hours.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 2000 units of FRETS-VWF73 activity per kilogram body weight of themammal (UFV73/kg). In a specific embodiment, the mammal is a human. Inone embodiment, the 20 UFV73 to 2000 UFV73/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 2000 UFV73/kg ADAMTS13is administered about twice a month. In one embodiment, the 20 to 2000UFV73/kg ADAMTS13 is administered about once a week. In one embodiment,the 20 to 2000 UFV73/kg ADAMTS13 is administered about twice a week. Inone embodiment, the 20 to 2000 UFV73/kg ADAMTS13 is administered aboutonce every 48 hours. In one embodiment, the 20 to 2000 UFV73/kg ADAMTS13is administered about once every 24 hours. In one embodiment, the 20 to2000 UFV73/kg ADAMTS13 is administered about once every 12 hours.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 1000 units of FRETS-VWF73 activity per kilogram body weight of themammal (UFV73/kg). In a specific embodiment, the mammal is a human. Inone embodiment, the 20 UFV73 to 1000 UFV73/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 1000 UFV73/kg ADAMTS13is administered about twice a month. In one embodiment, the 20 to 1000UFV73/kg ADAMTS13 is administered about once a week. In one embodiment,the 20 to 1000 UFV73/kg ADAMTS13 is administered about twice a week. Inone embodiment, the 20 to 1000 UFV73/kg ADAMTS13 is administered aboutonce every 48 hours. In one embodiment, the 20 to 1000 UFV73/kg ADAMTS13is administered about once every 24 hours. In one embodiment, the 20 to1000 UFV73/kg ADAMTS13 is administered about once every 12 hours.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 500 units of FRETS-VWF73 activity per kilogram body weight of themammal (UFV73/kg). In a specific embodiment, the mammal is a human. Inone embodiment, the 20 UFV73 to 500 UFV73/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 500 UFV73/kg ADAMTS13is administered about twice a month. In one embodiment, the 20 to 500UFV73/kg ADAMTS13 is administered about once a week. In one embodiment,the 20 to 500 UFV73/kg ADAMTS13 is administered about twice a week. Inone embodiment, the 20 to 500 UFV73/kg ADAMTS13 is administered aboutonce every 48 hours. In one embodiment, the 20 to 500 UFV73/kg ADAMTS13is administered about once every 24 hours. In one embodiment, the 20 to500 UFV73/kg ADAMTS13 is administered about once every 12 hours.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 200 units of FRETS-VWF73 activity per kilogram body weight of themammal (UFV73/kg). In a specific embodiment, the mammal is a human. Inone embodiment, the 20 UFV73 to 200 UFV73/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 200 UFV73/kg ADAMTS13is administered about twice a month. In one embodiment, the 20 to 200UFV73/kg ADAMTS13 is administered about once a week. In one embodiment,the 20 to 200 UFV73/kg ADAMTS13 is administered about twice a week. Inone embodiment, the 20 to 200 UFV73/kg ADAMTS13 is administered aboutonce every 48 hours. In one embodiment, the 20 to 200 UFV73/kg ADAMTS13is administered about once every 24 hours. In one embodiment, the 20 to200 UFV73/kg ADAMTS13 is administered about once every 12 hours.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 100 units of FRETS-VWF73 activity per kilogram body weight of themammal (UFV73/kg). In a specific embodiment, the mammal is a human. Inone embodiment, the 20 UFV73 to 100 UFV73/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 20 to 100 UFV73/kg ADAMTS13is administered about twice a month. In one embodiment, the 20 to 100UFV73/kg ADAMTS13 is administered about once a week. In one embodiment,the 20 to 100 UFV73/kg ADAMTS13 is administered about twice a week. Inone embodiment, the 20 to 100 UFV73/kg ADAMTS13 is administered aboutonce every 48 hours. In one embodiment, the 20 to 100 UFV73/kg ADAMTS13is administered about once every 24 hours. In one embodiment, the 20 to100 UFV73/kg ADAMTS13 is administered about once every 12 hours.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from40 to 200 units of FRETS-VWF73 activity per kilogram body weight of themammal (UFV73/kg). In a specific embodiment, the mammal is a human. Inone embodiment, the 40 UFV73 to 200 UFV73/kg ADAMTS13 is administeredabout once a month. In one embodiment, the 40 to 200 UFV73/kg ADAMTS13is administered about twice a month. In one embodiment, the 40 to 200UFV73/kg ADAMTS13 is administered about once a week. In one embodiment,the 40 to 200 UFV73/kg ADAMTS13 is administered about twice a week. Inone embodiment, the 40 to 200 UFV73/kg ADAMTS13 is administered aboutonce every 48 hours. In one embodiment, the 40 to 200 UFV73/kg ADAMTS13is administered about once every 24 hours. In one embodiment, the 40 to200 UFV73/kg ADAMTS13 is administered about once every 12 hours.

In one embodiment, the disclosure provides a method for prophylactictreatment of a mammal at risk for developing deep vein thrombosis, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 andfrequency of the dosing is selected from variations 1 to 1133 in Table2. In a specific embodiment, the mammal is a human.

Disseminated Intravascular Coagulation

In specific embodiments, an ADAMTS13 formulation described herein isused for the treatment and prophylaxis of disseminated intravascularcoagulation (DIC), specifically, sepsis-related DIC. DIC is a conditionin which blood clots form throughout the body's small blood vessels.These blood clots can reduce or block blood flow throughout the body andcan result in damage to tissues and organs. The blood clots in the smallblood vessels results from an increase in clotting activity. Thisincrease in activity over uses available platelets and clotting factors,thereby also increasing the chance of serious internal and externalbleeding by depleting the available source of platelets and clottingfactors. Accordingly, a patient with DIC will often suffer from bloodclots and severe bleeding disorders.

Certain diseases such as sepsis, surgery/trauma, cancer, complicationsof childbirth/pregnancy, venomous snake bites (rattlesnakes and vipers),frostbite, and burns can cause clotting factors to become overactive andcan lead to DIC. DIC can also be acute (developing quickly over hours ordays) or chronic (developing over weeks or months). While both types ofDIC require medical treatment, acute DIC must be treated immediately toprevent excessive blood clotting in the small blood vessels that quicklylead to severe bleeding. Accordingly, it is important to have apharmaceutical composition that can be quickly and easily administeredto treat DIC, especially acute DIC such as sepsis-related DIC. Thus, itwould be beneficial to develop a subcutaneous ADAMTS13 formulation and amethod of ADAMTS13 subcutaneous delivery.

In some embodiments, the pharmaceutical composition is administeredimmediately upon discovery of disseminated intravascular coagulation,e.g., within 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes,30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes,60 minutes, 90 minutes, 110 minutes, 120 minutes, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 or more hours, or anycombination thereof. Accordingly, it is important to have apharmaceutical composition that can be quickly and easily administered.

In one embodiment, the disclosure provides a method for treatingdisseminated intravascular coagulation in a mammal in need thereof, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 4000 units of FRETS-VWF73 activity per kilogram body weight of themammal (U_(FV73)/kg). In a specific embodiment, the mammal is a human.In one embodiment, the 20 U_(FV73) to 4000 U_(FV73)/kg ADAMTS13 isadministered within 10 minutes of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to4000 U_(FV73)/kg ADAMTS13 is administered within 30 minutes of discoveryof disseminated intravascular coagulation in the mammal. In oneembodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 is administered within60 minutes of discovery of disseminated intravascular coagulation in themammal. In one embodiment, the 20 to 4000 U_(FV73)/kg ADAMTS13 isadministered within 4 hours of discovery of disseminated intravascularcoagulation in the mammal. In one embodiment, the 20 to 4000 U_(FV73)/kgADAMTS13 is administered within 12 hours of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to4000 U_(FV73)/kg ADAMTS13 is administered within 24 hours of discoveryof disseminated intravascular coagulation in the mammal.

In one embodiment, the disclosure provides a method for treatingdisseminated intravascular coagulation in a mammal in need thereof, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 2000 units of FRETS-VWF73 activity per kilogram body weight of themammal (U_(FV73)/kg). In a specific embodiment, the mammal is a human.In one embodiment, the 20 U_(FV73) to 2000 U_(FV73)/kg ADAMTS13 isadministered within 10 minutes of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to2000 U_(FV73)/kg ADAMTS13 is administered within 30 minutes of discoveryof disseminated intravascular coagulation in the mammal. In oneembodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 is administered within60 minutes of discovery of disseminated intravascular coagulation in themammal. In one embodiment, the 20 to 2000 U_(FV73)/kg ADAMTS13 isadministered within 4 hours of discovery of disseminated intravascularcoagulation in the mammal. In one embodiment, the 20 to 2000 U_(FV73)/kgADAMTS13 is administered within 12 hours of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to2000 U_(FV73)/kg ADAMTS13 is administered within 24 hours of discoveryof disseminated intravascular coagulation in the mammal.

In one embodiment, the disclosure provides a method for treatingdisseminated intravascular coagulation in a mammal in need thereof, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 1000 units of FRETS-VWF73 activity per kilogram body weight of themammal (U_(FV73)/kg). In a specific embodiment, the mammal is a human.In one embodiment, the 20 U_(FV73) to 1000 U_(FV73)/kg ADAMTS13 isadministered within 10 minutes of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to1000 U_(FV73)/kg ADAMTS13 is administered within 30 minutes of discoveryof disseminated intravascular coagulation in the mammal. In oneembodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 is administered within60 minutes of discovery of disseminated intravascular coagulation in themammal. In one embodiment, the 20 to 1000 U_(FV73)/kg ADAMTS13 isadministered within 4 hours of discovery of disseminated intravascularcoagulation in the mammal. In one embodiment, the 20 to 1000 U_(FV73)/kgADAMTS13 is administered within 12 hours of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to1000 U_(FV73)/kg ADAMTS13 is administered within 24 hours of discoveryof disseminated intravascular coagulation in the mammal.

In one embodiment, the disclosure provides a method for treatingdisseminated intravascular coagulation in a mammal in need thereof, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 500 units of FRETS-VWF73 activity per kilogram body weight of themammal (U_(FV73)/kg). In a specific embodiment, the mammal is a human.In one embodiment, the 20 U_(FV73) to 500 U_(FV73)/kg ADAMTS13 isadministered within 10 minutes of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to500 U_(FV73)/kg ADAMTS13 is administered within 30 minutes of discoveryof disseminated intravascular coagulation in the mammal. In oneembodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 is administered within 60minutes of discovery of disseminated intravascular coagulation in themammal. In one embodiment, the 20 to 500 U_(FV73)/kg ADAMTS13 isadministered within 4 hours of discovery of disseminated intravascularcoagulation in the mammal. In one embodiment, the 20 to 500 U_(FV73)/kgADAMTS13 is administered within 12 hours of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to500 U_(FV73)/kg ADAMTS13 is administered within 24 hours of discovery ofdisseminated intravascular coagulation in the mammal.

In one embodiment, the disclosure provides a method for treatingdisseminated intravascular coagulation in a mammal in need thereof, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 200 units of FRETS-VWF73 activity per kilogram body weight of themammal (U_(FV73)/kg). In a specific embodiment, the mammal is a human.In one embodiment, the 20 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 isadministered within 10 minutes of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to200 U_(FV73)/kg ADAMTS13 is administered within 30 minutes of discoveryof disseminated intravascular coagulation in the mammal. In oneembodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 is administered within 60minutes of discovery of disseminated intravascular coagulation in themammal. In one embodiment, the 20 to 200 U_(FV73)/kg ADAMTS13 isadministered within 4 hours of discovery of disseminated intravascularcoagulation in the mammal. In one embodiment, the 20 to 200 U_(FV73)/kgADAMTS13 is administered within 12 hours of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to200 U_(FV73)/kg ADAMTS13 is administered within 24 hours of discovery ofdisseminated intravascular coagulation in the mammal.

In one embodiment, the disclosure provides a method for treatingdisseminated intravascular coagulation in a mammal in need thereof, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from20 to 100 units of FRETS-VWF73 activity per kilogram body weight of themammal (U_(FV73)/kg). In a specific embodiment, the mammal is a human.In one embodiment, the 20 U_(FV73) to 100 U_(FV73)/kg ADAMTS13 isadministered within 10 minutes of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to100 U_(FV73)/kg ADAMTS13 is administered within 30 minutes of discoveryof disseminated intravascular coagulation in the mammal. In oneembodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 is administered within 60minutes of discovery of disseminated intravascular coagulation in themammal. In one embodiment, the 20 to 100 U_(FV73)/kg ADAMTS13 isadministered within 4 hours of discovery of disseminated intravascularcoagulation in the mammal. In one embodiment, the 20 to 100 U_(FV73)/kgADAMTS13 is administered within 12 hours of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 20 to100 U_(FV73)/kg ADAMTS13 is administered within 24 hours of discovery ofdisseminated intravascular coagulation in the mammal.

In one embodiment, the disclosure provides a method for treatingdisseminated intravascular coagulation in a mammal in need thereof, themethod including subcutaneously administering a therapeuticallyeffective amount of a composition comprising isolated ADAMTS13 to themammal, where the therapeutically effective amount of ADAMTS13 is from40 to 200 units of FRETS-VWF73 activity per kilogram body weight of themammal (U_(FV73)/kg). In a specific embodiment, the mammal is a human.In one embodiment, the 40 U_(FV73) to 200 U_(FV73)/kg ADAMTS13 isadministered within 10 minutes of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 40 to200 U_(FV73)/kg ADAMTS13 is administered within 30 minutes of discoveryof disseminated intravascular coagulation in the mammal. In oneembodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 is administered within 60minutes of discovery of disseminated intravascular coagulation in themammal. In one embodiment, the 40 to 200 U_(FV73)/kg ADAMTS13 isadministered within 4 hours of discovery of disseminated intravascularcoagulation in the mammal. In one embodiment, the 40 to 200 U_(FV73)/kgADAMTS13 is administered within 12 hours of discovery of disseminatedintravascular coagulation in the mammal. In one embodiment, the 40 to200 U_(FV73)/kg ADAMTS13 is administered within 24 hours of discovery ofdisseminated intravascular coagulation in the mammal.

V. ADAMTS13 Kits

In another aspect, kits are provided for the treatment of a disease orcondition associated with ADAMTS13 or VWF dysfunction. In oneembodiment, the kit comprises a formulation of rADAMTS13. In someembodiments, the kits provided herein may contain one or more dose of aliquid or lyophilized formulation as provided herein. When the kitscomprise a lyophilized rADAMTS13 formulation, generally the kits willalso contain a suitable liquid for reconstitution of the liquidformulation, for example, sterile water or a pharmaceutically acceptablebuffer. In some embodiments, a kit includes an ADAMTS13 formulationprepackaged in a syringe for subcutaneous administration by a healthcare professional or for home use.

In one embodiment, a kit is provided comprising between about 10 unitsof FRETS-VWF73 activity and about 10,000 units of FRETS-VWF73 activity.In other embodiments, the kit may provide, for example, between about 20units of FRETS-VWF73 (U_(FV73)) activity and about 8,000 units ofFRETS-VWF73 activity, or between about 30 U_(FV73) and about 6,000U_(FV73), or between about 40 U_(FV73) and about 4,000 U_(FV73), orbetween about 50 U_(FV73) and about 3,000 U_(FV73), or between about 75U_(FV73) and about 2,500 U_(FV73), or between about 100 U_(FV73) andabout 2,000 U_(FV73), or between about 200 U_(FV73) and about 1,500U_(FV73), or between about other ranges therein. In certain embodiments,a kit may provide about 10 units of FRETS-VWF73 activity, or about 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600,2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600,3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600,4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600,5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600,6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600,7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600,8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600,9,700, 9,800, 9,900, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000,16,000, 17,000, 18,000, 19,000, 20,000 or more units of FRETS-VWF73activity.

In certain embodiments, the kit is for a single administration or doseof ADAMTS13. In other embodiments, the kit may contain multiple doses ofADAMTS13 for subcutaneous administration. In one embodiment, the kit maycomprise an ADAMTS13 formulation prepackaged in a syringe forsubcutaneous administration by a health care professional or for homeuse.

VI. EXAMPLES Example 1 The Pharmacokinetic Properties of rADAMTS13 withIntravenous and Subcutaneous Administration

This study was conducted to evaluate the pharmacokinetic properties ofrecombinant ADAMTS13 in Göttingen minipigs after intravenous (i.v.) orsubcutaneous (s.c.) administration. In this study, 4 male Göttingenminipigs were administered i.v. ADMTS13 (the “intravenous group”) and 4male Göttingen minipigs were administered s.c. ADMTS13 (the“subcutaneous group”).

Male Göttingen minipigs were trained before the start of the study tocooperate with the study procedures. Thus, the animals were notrestrained during administration of anesthetics or the test item andblood sampling.

The animals were anesthetized for catheter implantation using a Zoletilmix consisting of 1.11 mg/kg tiletamine, 1.11 mg/kg zolazepam, 0.56mg/kg xylazine, 0.56 mg/kg ketamine, and 0.11 mg/kg butorphanole (i.m.).Isoflurane was used for anesthesia maintenance when necessary. Theanimals received pure oxygen via a mask or an endotracheal tube duringsurgery. The jugular region was shaved and disinfected. A centralcatheter was inserted into the cranial V. cava using the Seldingertechnique. 4 mg/kg carprofene was administered for analgesia beforerecovery from anesthesia.

The intravenous group received a nominal dose of 200 FRETS-U/kgrADAMTS13 i.v. (0.085 mL/kg). The subcutaneous group received a nominaldose of 1000 FRETS-U/kg rADAMTS13s.c. (0.456 mL/kg). Blood was sampledvia the central venous catheter before administration of the test itemand 5 min, 1 h, 3 h, 6 h, 9 h. 24 h, 32 h, 48 h, 56 h and 72 h afteradministration of the test item. Blood samples were prepared as follows:the first 0.5-0.7 mL were discarded to avoid dilution with the catheterlock solution (saline). Then, 0.8 mL blood was collected and mixed with0.2 mL sodium citrate. The blood sample was centrifuged with 5700 rpm(1×10 min, 1×5 min). The plasma was stored at <−60° C.

ADAMTS13 activity was determined by a FRETS assay and antigen by anELISA. Furthermore, the potential development of binding antibodiesagainst human rADAMTS13 and the influence of rADAMTS13 on the multimerpattern of endogenous VWF were assessed.

As expected, after i.v. administration the median T_(max) (time toC_(max)) was 5 min, indicating immediate bioavailability. Median T_(max)after s.c. administration was 24 h for ADAMTS13 activity and 28 h forADAMTS13 antigen. Mean plasma concentrations of ADAMTS13 activity aresummarized in FIG. 1 and the averaged concentrations of ADAMTS13 antigenare summarized in FIG. 2.

The dose-adjusted AUC_(0-tlast) [h*U/mL/U/kg and h*μg/mL/μg/kg] afteri.v. administration was 0.306 for activity and 0.373 for antigen.AUC_(0-tlast) was computed using the trapezoidal rule from time point 0to the last sampling time (t_(last)) (Hauschke et al. 2007). Thedose-adjusted AUC_(0-tlast) following s.c. administration was 0.198 foractivity and 0.243 for antigen. Thus, the ratio of the dose-adjustedAUC_(0-tlast) following s.c. and i.v. administration was 0.646 foractivity and 0.651 for antigen. IVR [%] was 55.8 (activity) and 79.3(antigen) after i.v. administration and 13.8 (activity) and 17.3(antigen) after s.c. administration.

Since the plasma level of rADAMTS13 did not decline sufficiently duringthe observation period, the estimation of the terminal and initialhalf-lives has to be viewed with caution. Thus, the dependent parameters(MRT, Vss, Cls, AUC_(0-inf)) may be unreliable as well. A follow-upstudy will be conducted to assess these parameters. Assessment ofbinding antibodies against rADAMTS13 was negative for all samples fromall animals.

Results of the assessment of the minipigs' VWF multimer pattern wereindicative for a very limited cleavage of endogenous VWF by recombinantADAMTS13 (Table 3). The satellite band close to a low VWF-mer slightlyincreased in intensity over time in animals receiving s.c.administration, but not in animals receiving i.v. administration. Theheight of the VWF multimer patterns did not change in either group.

TABLE 3 Multimer analysis of recombinant ADAMTS13 from low and highresolution agarose gels. Experimental No. of Group animals VWF cleavageIntravenous 1 No Group 2 No (200 IU/kg i.v.) 3 No 4 No Subcutaneous 1Slight increase in one satellite band intensity Group 2 Slight increasein one satellite band intensity (1000 IU/kg s.c.) 3 Slight increase inone satellite band intensity 4 Slight increase in one satellite bandintensity

Example 2 The Pharmacokinetic Properties of rADAMTS13 with Intravenousand Subcutaneous Administration

This study was conducted to evaluate the pharmacokinetic properties ofrecombinant ADAMTS13 in Göttingen minipigs after intravenous (i.v.) orsubcutaneous (s.c.) administration. In this study, 4 male Göttingenminipigs were administered i.v. ADMTS13 (the “intravenous group”) and 4male Göttingen minipigs were administered s.c. ADMTS13 (the“subcutaneous group”).

The male Göttingen minipigs were acclimated to study procedures andanesthetized for catheter implantation as described in Example 1.

This study was a trial using 3 and 5 male Göttingen minipigs per group.Both groups received a nominal dose of 200 FRETS-U/kg rADAMTS13. Threeanimals were dosed i.v., the other five animals were dosed s.c. Bloodwas sampled via the central venous catheter before administration of thetest item and 5 min, 1 h, 2 h, 6 h, 5 h, 23 h, 30 h, 47 h, 54 h, 71 h,78 h, 95 h and 102 h after administration of the test item.

Blood samples were prepared as follows: The first 0.5-0.7 mL werediscarded to avoid dilution with the catheter lock solution (saline).Then, 0.8 mL blood was collected and mixed with 0.2 mL sodium citrate.The blood sample was centrifuged with 5700 rpm (1×10 min, 1×5 min). Theplasma was stored at <−60° C.

ADAMTS13 activity was determined by a FRETS assay and antigen by anELISA.

As expected, after i.v. administration the median T_(max) (time toC_(max)) was 5 min, indicating immediate bioavailability. Median T_(max)after s.c. administration was 30 h. Mean plasma concentrations ofADAMTS13 activity are summarized in FIG. 3 and the averagedconcentrations of ADAMTS13 antigen are summarized in FIG. 4.

The AUC_(0-tlast) [U/mL/*h and μg/mL*h] after i.v. administration was68.25 for activity and 39.81 for antigen. The AUC_(0-tlast) followings.c. administration was 47.16 for activity and 26.23 for antigen. IVR[%] was 61.0 (activity) and 45.6 (antigen) after i.v. administration and13.0 (activity) and 9.6 (antigen) after s.c. administration.

Terminal half-life [h] after i.v. administration was 46.96 for activityand 48.83 for antigen. Terminal half-life after s.c. administration was56.76 for activity and 41.25 for antigen. MRT [h] was 64.08 (activity)and 66.26 (antigen) after i.v. administration and 104.2 (activity) and77.52 (antigen) after s.c. administration.

In summary, the bioavailability of the s.c. administration relative tothe i.v. administration was 65.9% and 69.1% for ADAMTS13 antigen andADAMTS13 activity, respectively.

What is claimed is:
 1. A method for treating a blood clotting disorderin a mammal, the method comprising subcutaneously administering atherapeutically effective amount of a composition comprising isolatedADAMTS13 to the mammal in need thereof, wherein the therapeuticallyeffective amount of ADAMTS13 is from 20 to 4,000 activity units perkilogram body weight, and the bioavailability of the ADAMTS13 aftersubcutaneous administration is 50-80% as compared to intravenousadministration normalized for the same dose, and thereby treating theblood clotting disorder in the mammal.
 2. The method of claim 1, whereinthe blood clotting disorder is selected from the group consisting ofinherited TTP, acquired TTP, cerebral infarction, myocardial infarction,ischemic/reperfusion injury, deep vein thrombosis, and sepsis-relateddisseminated intravasular coagulation.
 3. The method of claim 1, whereinthe bioavailability of the ADAMTS13 after subcutaneous administration is55-80% as compared to intravenous administration normalized for the samedose.
 4. The method of claim 1, wherein the bioavailability of theADAMTS13 after subcutaneous administration is 60-80% as compared tointravenous administration normalized for the same dose.
 5. The methodof claim 1, wherein the bioavailability of the ADAMTS13 aftersubcutaneous administration is 65-80% as compared to intravenousadministration normalized for the same dose.
 6. The method of claim 1,wherein the bioavailability of the ADAMTS13 after subcutaneousadministration is 70-80% as compared to intravenous administrationnormalized for the same dose.
 7. The method of claim 1, wherein thebioavailability of the ADAMTS13 after subcutaneous administration is75-80% as compared to intravenous administration normalized for the samedose.
 8. The method of claim 2, wherein the blood clotting disorder isinherited TTP.
 9. The method of claim 8, wherein the therapeuticallyeffective amount is from 20 to 160 activity units per kilogram bodyweight.
 10. The method of claim 2, wherein the blood clotting disorderis acquired TTP.
 11. The method of claim 10, wherein the therapeuticallyeffective amount is from 40 to 2,000 activity units per kilogram bodyweight.
 12. The method of claim 2, wherein the blood clotting disorderis cerebral infarction or ischemia reperfusion injury.
 13. The method ofclaim 12, wherein the therapeutically effective amount is from 40 to4,000 activity units per kilogram body weight.
 14. The method of claim2, wherein the blood clotting disorder is myocardial infarction orischemia reperfusion injury.
 15. The method of claim 14, wherein thetherapeutically effective amount is from 40 to 2,000 activity units perkilogram body weight.
 16. The method of claim 1, wherein the ADAMTS13 isadministered in a single bolus injection, monthly, every two weeks,weekly, twice a week, daily, every 12 hours, every 8 hours, every sixhours, every four hours, or every two hours.
 17. The method of claim 1,wherein the ADAMTS13 is recombinant.
 18. The method of claim 1, whereinthe ADAMTS13 is plasma derived.
 19. The method of claim 1, wherein themammal is a human.
 20. The method of claim 1, wherein the composition isa stable aqueous solution ready for administration.
 21. The method ofclaim 1, wherein the composition is lyophilized.
 22. The method of claim21, wherein the composition is reconstituted with a pharmaceuticallyacceptable vehicle suitable for injection.